Multi-specific binding proteins that bind cd33, nkg2d, and cd16, and methods of use

ABSTRACT

Multi-specific binding proteins that bind to and kill human cancer cells expressing CD33 (Siglec-3) are described, as well as pharmaceutical compositions and therapeutic methods useful for the treatment of CD33 expressing cancer. The invention relates to multi-specific binding proteins that bind to human cancer cells expressing CD33, and exhibit high potency and maximum lysis of target cells compared to anti-CD33 monoclonal antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/971,104 filed on Aug. 19, 2020, pending, which is a U.S. NationalStage Application of International Patent Application No.PCT/US2019/018751, filed Feb. 20, 2019, which claims the benefit of andpriority to U.S. Provisional Patent Application No. 62/632,756, filedFeb. 20, 2018, the disclosure of which is hereby incorporated byreference in its entirety for all purposes; and U.S. Provisional PatentApplication No. 62/677,137, filed May 28, 2018.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 25, 2022, isnamed 14247-659-999_Sequence_Lisitng.txt and is 571,280 bytes in size.

FIELD OF THE INVENTION

The invention relates to multi-specific binding proteins that bind toCD33 (Siglec-3), and exhibit high potency and maximum lysis of targetcells compared to anti-CD33 monoclonal antibodies.

BACKGROUND

Cancer continues to be a significant health problem despite thesubstantial research efforts and scientific advances reported in theliterature for treating this disease. Some of the most frequentlydiagnosed cancers in adults include prostate cancer, breast cancer, andlung cancer. Hematological malignancies, though less frequent than solidcancers, have low survival rates. Current treatment options for thesecancers are not effective for all patients and/or can have substantialadverse side effects. Other types of cancer also remain challenging totreat using existing therapeutic options.

Cancer immunotherapies are desirable because they are highly specificand can facilitate destruction of cancer cells using the patient's ownimmune system. Fusion proteins such as bi-specific T-cell engagers arecancer immunotherapies described in the literature that bind to tumorcells and T-cells to facilitate destruction of tumor cells. T cells aremajor effectors of the adaptive immune system that attack foreign cellsas well as host cells that present mutant or mis-expressed peptides.Cells targeted by T cells may be virally-infected, such that theyexpress foreign proteins, or malignant, where they might express mutantproteins. T cells recognize target cells via their T cell receptor (TCR)engaging intracellular peptides presented by major histocompatibilitycomplex proteins on target cells. Individual T cells typically recognizetarget cells bearing specific MHC-peptide complexes, but novel agentshave been developed that usurp and amplify this natural process fortherapeutic benefit. Bi-specific T cell engagers link antigen bindingsite(s) of tumor-associated antigens to antigen binding site(s) ofcomponents of the TCR complex to redirect T cell activity towardsdesired target cells independent of native peptide-MHC recognition. Forexample, Blincyto is an FDA-approved T cell engager that targets CD19 onmalignant B cells.

T cells can also be engineered to express chimeric antigen receptors(CAR) that endow it with target recognition capabilities of its CAR.CARs contain antigen binding site(s) to tumor associated antigens linkedto T cell activation domains. These CAR-T cells can also be employed totarget malignant cells, and some have been FDA-approved for use againstB cell malignancies.

Antibodies that bind to certain tumor-associated antigens and to certainimmune cells have been described in the literature. See, e.g., WO2016/134371 and WO 2015/095412. Antibody-drug conjugates orimmunocytokines using antigen binding sites targeting tumor associatedantigens to deliver toxic agents or immune-modulatory cytokines tospecific target cells.

Natural killer (NK) cells are a component of the innate immune systemand make up approximately 15% of circulating lymphocytes. NK cellsinfiltrate virtually all tissues and were originally characterized bytheir ability to kill tumor cells effectively without the need for priorsensitization. Activated NK cells kill target cells by means similar tocytotoxic T cells—i.e., via cytolytic granules that contain perforin andgranzymes as well as via death receptor pathways. Activated NK cellsalso secrete inflammatory cytokines such as IFN-gamma and chemokinesthat promote the recruitment of other leukocytes to the target tissue.

NK cells respond to signals through a variety of activating andinhibitory receptors on their surface. For example, when NK cellsencounter healthy self-cells, their activity is inhibited throughactivation of the killer-cell immunoglobulin-like receptors (KIRs).Alternatively, when NK cells encounter foreign cells or cancer cells,they are activated via their activating receptors (e.g., NKG2D, NCRs,DNAM1). NK cells are also activated by the constant region of someimmunoglobulins through CD16 receptors on their surface. The overallsensitivity of NK cells to activation depends on the sum of stimulatoryand inhibitory signals.

CD33 is a member of the sialic acid-binding immunoglobulin-like lectins.As a transmembrane receptor mainly expressed on cells of myeloidlineage, CD33 modulates inflammatory and immune responses through adampening effect on tyrosine kinase-driven signaling pathways. Forexample, CD33 was shown to constitutively suppress the production ofpro-inflammatory cytokines such as IL-1β, TNF-α, and IL-8 by humanmonocytes.

CD33 is associated with hematopoietic cancers. It is broadly expressedin blasts of nearly all acute myeloid leukemia (AML). Furthermore,hematopoietic cancer stem and/or progenitor cells are found to be CD33⁺,implying that CD33-directed therapy could potentially eradicatemalignant stem and/or progenitor cells in such cases while sparingnormal hematopoietic stem cells. In addition to its expression in AML,CD33 is found on other myeloid neoplasms (e.g., myelodysplasticsyndromes and myeloproliferative neoplasms) and on subsets of B-cell andT-cell acute lymphoblastic leukemias (ALL)/lymphoblastic lymphomas. Thisexpression pattern has led to the use of CD33-directed therapeutics inpatients with malignancies including AML, myelodysplastic syndromes,chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloidleukemia, and ALLs.

SUMMARY OF THE INVENTION

The invention provides multi-specific binding proteins that bind to CD33on a cancer cell and to the NKG2D receptor and CD16 receptor on naturalkiller cells. Such proteins can engage more than one kind of NKactivating receptor, and may block the binding of natural ligands toNKG2D. In certain embodiments, the proteins can agonize NK cells inhumans, and in other species such as rodents and cynomolgus monkeys.Various aspects and embodiments of the invention are described infurther detail below.

In certain embodiments, the present invention provides a protein thatincludes a human CD33 antigen-binding site including a heavy chainvariable domain, which includes an amino acid sequence at least 90%(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, 294, 296, 298, 300, or 302, and further comprises asecond antigen binding site same or different from the antigen-bindingsite that binds to human CD33.

In certain embodiments, the present invention provides anantigen-binding site in a protein (e.g., a multi-specific bindingprotein) that binds to CD33 on a cancer cell, and the NKG2D receptor andCD16 receptor on natural killer cells to activate the natural killercell. The binding protein (e.g., a multi-specific binding protein) isuseful in the pharmaceutical compositions and therapeutic methodsdescribed herein. Binding of the protein including an antigen-bindingsite that binds to CD33, and to NKG2D receptor and CD16 receptor onnatural killer cell enhances the activity of the natural killer celltoward destruction of a cancer cell. Binding of the protein including anantigen-binding site that binds to CD33 (e.g., a multi-specific bindingprotein) on a cancer cell brings the cancer cell into proximity to thenatural killer cell, which facilitates direct and indirect destructionof the cancer cell by the natural killer cell. Further description ofexemplary multi-specific binding proteins is provided below.

The first component of the multi-specific binding proteins of thepresent disclosure binds to CD33-expressing cells, which can include butare not limited to AML, myelodysplastic syndromes, chronicmyelomonocytic leukemia, myeloid blast crisis of chronic myeloidleukemia, and ALLs.

The second component of the multi-specific binding proteins of thepresent disclosure binds to NKG2D receptor-expressing cells, which caninclude but are not limited to NK cells, γδ T cells and CD8⁺αβ T cells.Upon NKG2D binding, the multi-specific binding proteins may blocknatural ligands, such as ULBP6 and MICA, from binding to NKG2D andactivating NKG2D receptors. The NKG2D antigen binding site can include,for example:

(1) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:81 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:82[ADI-29379];

(2) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:83 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:84[ADI-29463];

(3) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:85 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:86[ADI-27744];

(4) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:87 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:88[ADI-27749];

(5) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:191 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:88[A49MI]; or

(6) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:89 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:90[ADI-29378].

The third component for the multi-specific binding proteins of thepresent disclosure binds to cells expressing CD16, an Fc receptor on thesurface of leukocytes including natural killer cells, macrophages,neutrophils, eosinophils, mast cells, and follicular dendritic cells.

In certain embodiments, any of the foregoing isolated antibodies has aK_(D) of 1 nM or lower, 5 nM or lower, or 12 nM or lower forextracellular domain of human CD33, as measured by surface plasmonresonance (SPR) (e.g., using the Biacore method described in Example 1infra) or by bio-layer interferometry (BLI) (e.g., using the Octetmethod described in Example 1 infra), and/or binds CD33 from a bodyfluid, tissue, and/or cell of a subject. In certain embodiments, any ofthe foregoing isolated antibodies has a K_(d) (i.e., off-rate, alsocalled K_(off)) equal to or lower than 1×10⁻⁵, 1×10⁻⁴, 1×10⁻³, 5×10⁻³,0.01, 0.02, or 0.05 1/s, as measured by SPR (e.g., using the Biacoremethod described in Example 1 infra) or by BLI (e.g., using the Octetmethod described in Example 1 infra).

Some proteins of the present disclosure bind to NKG2D with a K_(D) of 10nM or weaker affinity.

In another aspect, the invention provides one or more isolated nucleicacids comprising sequences encoding an immunoglobulin heavy chain and/orimmunoglobulin light chain variable region of any one of the foregoingantibodies. The invention provides one or more expression vectors thatexpress the immunoglobulin heavy chain and/or immunoglobulin light chainvariable region of any one of the foregoing antibodies. Similarly theinvention provides host cells comprising one or more of the foregoingexpression vectors and/or isolated nucleic acids.

Formulations including any of the proteins that include a CD33-bindingdomain described herein and methods of enhancing tumor cell death usingthese proteins and/or formulations are also provided.

In another aspect, the invention provides a method of treating a cancer,for example, a CD33-associated cancer, in a subject. The methodcomprises administering to the subject an effective amount of a proteincontaining any CD33-binding domain described herein, for example, amulti-specific protein containing a CD33binding domain, an NKG2D-bindingdomain and a CD16-binding domain, to treat the cancer in the subject.

In another aspect, the invention provides a method of inhibiting cancergrowth, for example, the growth of a CD33-associated cancer, in asubject. The method comprises exposing the subject to an effectiveamount of an antibody comprising any CD33-binding domain describedherein, for example, a multi-specific protein containing an CD33-bindingdomain, an NKG2D-binding domain and a CD16-binding domain, to inhibitcancer growth in the subject.

Another aspect of the invention provides a method of treating cancer ina patient. The method comprises administering to a patient in needthereof a therapeutically effective amount of the multi-specific bindingprotein described herein. Exemplary cancers for treatment using themulti-specific binding proteins include, for example, wherein the canceris selected from the group consisting of AML, myelodysplastic syndromes,chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloidleukemia, and ALLs.

Another aspect of the invention provides a protein comprising:

(a) a first antigen-binding site comprising an Fab fragment that bindsNKG2D;(b) a second antigen-binding site comprising a single-chain variablefragment (scFv) that binds CD33 comprising a heavy chain variable domainand a light chain variable domain; and(c) an antibody Fc domain or a portion thereof sufficient to bind CD16,or a third antigen-binding site that binds CD16, wherein the scFv islinked to the antibody Fc domain or a portion thereof sufficient to bindCD16, or the third antigen-binding site that binds CD16, via a hingecomprising Ala-Ser or Gly-Ala-Ser.

In certain embodiments, the scFv is linked to the antibody Fc domain. Incertain embodiments, the heavy chain variable domain of the scFv forms adisulfide bridge with the light chain variable domain of the scFv. Incertain embodiments, the disulfide bridge is formed between C44 from theheavy chain variable domain and C100 from the light chain variabledomain.

In certain embodiments, the scFv is linked to the antibody Fc domain,wherein the light chain variable domain of the scFv is positioned at theN-terminus of the heavy chain variable domain of the scFv, and is linkedto the heavy chain variable domain of the scFv via a flexible linker(GlyGlyGlyGlySer)4 ((G4S)₄), and the Fab is linked to the antibody Fcdomain.

In certain embodiments, the present invention provides a protein asdisclosed herein, wherein the heavy chain variable domain of the scFv islinked to the light chain variable domain of the scFv via a flexiblelinker. In certain embodiments, the flexible linker comprises(GlyGlyGlyGlySer)4 ((G4S)₄).

In certain embodiments, the present invention provides a protein asdisclosed herein, wherein the heavy chain variable domain of the scFv ispositioned at the N-terminus or the C-terminus of the light chainvariable domain of the scFv. In certain embodiments, the light chainvariable domain of the scFv is positioned at the N-terminus of the heavychain variable domain of the scFv.

In certain embodiments, the present invention provides a protein asdisclosed herein, wherein the Fab fragment is linked to the antibody Fcdomain or a portion thereof sufficient to bind CD16 or the thirdantigen-binding site that binds CD16. In certain embodiments, the heavychain portion of the Fab fragment comprises a heavy chain variabledomain and a CH1 domain, and wherein the heavy chain variable domain islinked to the CH1 domain. In certain embodiments, the Fab fragment islinked to the antibody Fc domain.

In certain embodiments, the first antigen-binding site that binds NKG2Dcomprises a heavy chain variable domain comprisingcomplementarity-determining region 1 (CDR1), complementarity-determiningregion 2 (CDR2), and complementarity-determining region 3 (CDR3)sequences represented by the amino acid sequences of SEQ ID NOs:93, 94,and 95, respectively; and a light chain variable domain comprising CDR1,CDR2, and CDR3 sequences represented by the amino acid sequences of SEQID NOs:96, 97, and 98, respectively. In certain embodiments, the firstantigen-binding site that binds NKG2D comprises a heavy chain variabledomain comprising CDR1, CDR2, and CDR3 sequences represented by theamino acid sequences of SEQ ID NOs:99, 100, and 101, respectively; and alight chain variable domain comprising CDR1, CDR2, and CDR3 sequencesrepresented by the amino acid sequences of SEQ ID NOs:102, 103, and 104,respectively. In certain embodiments, the first antigen-binding sitethat binds NKG2D comprises a heavy chain variable domain comprisingCDR1, CDR2, and CDR3 sequences represented by the amino acid sequencesof SEQ ID NOs:105, 106, and 107, respectively; and a light chainvariable domain comprising CDR1, CDR2, and CDR3 sequences represented bythe amino acid sequences of SEQ ID NOs:108, 109, and 110, respectively.In certain embodiments, the first antigen-binding site that binds NKG2Dcomprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3sequences represented by the amino acid sequences of SEQ ID NOs:192,112, and 193, respectively; and a light chain variable domain comprisingCDR1, CDR2, and CDR3 sequences represented by the amino acid sequencesof SEQ ID NOs:114, 115, and 116, respectively. In certain embodiments,the first antigen-binding site that binds NKG2D comprises a heavy chainvariable domain comprising CDR1, CDR2, and CDR3 sequences represented bythe amino acid sequences of SEQ ID NOs:192, 112, and 195, respectively;and a light chain variable domain comprising CDR1, CDR2, and CDR3sequences represented by the amino acid sequences of SEQ ID NOs:114,115, and 116, respectively. In certain embodiments, the firstantigen-binding site that binds NKG2D comprises a heavy chain variabledomain comprising CDR1, CDR2, and CDR3 sequences represented by theamino acid sequences of SEQ ID NOs:117, 118, and 119, respectively; anda light chain variable domain comprising CDR1, CDR2, and CDR3 sequencesrepresented by the amino acid sequences of SEQ ID NOs:120, 121, and 122,respectively.

In certain embodiments, the first antigen-binding site that binds NKG2Dcomprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:81 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:82.In certain embodiments, the first antigen-binding site that binds NKG2Dcomprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:83 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:84.In certain embodiments, the first antigen-binding site that binds NKG2Dcomprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:85 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:86.In certain embodiments, the first antigen-binding site that binds NKG2Dcomprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:87 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:88.In certain embodiments, the first antigen-binding site that binds NKG2Dcomprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:191 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:88.In certain embodiments, the first antigen-binding site that binds NKG2Dcomprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:89 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:90.

In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3sequences represented by the amino acid sequences of SEQ ID NOs:181, 46,and 182, respectively; and a light chain variable domain comprisingCDR1, CDR2, and CDR3 sequences represented by the amino acid sequencesof SEQ ID NOs:48, 49, and 50, respectively. In certain embodiments, thesecond antigen-binding site that binds CD33 comprises a heavy chainvariable domain comprising CDR1, CDR2, and CDR3 sequences represented bythe amino acid sequences of SEQ ID NOs:183, 34, and 184, respectively;and a light chain variable domain comprising CDR1, CDR2, and CDR3sequences represented by the amino acid sequences of SEQ ID NOs:36, 185,and 38, respectively.

In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:9 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:10. Incertain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:5 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:6. Incertain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:1 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:2. Incertain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:3 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:4. Incertain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:7 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8. Incertain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:11 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:12.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:13 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:14.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:15 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:16.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:17 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:18.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:19 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:20.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:266 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:267.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:268 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:269.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:270 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:271.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:272 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:273.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:274 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:275.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:276 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:277.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:278 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:279.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:280 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:281.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:282 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:283.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:284 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:285.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:286 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:287.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:288 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:289.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:290 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:291.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:292 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:293.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:294 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:295.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:296 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:297.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:298 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:299.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:300 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:301.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:302 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:303.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:418 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:419.In certain embodiments, the second antigen-binding site that binds CD33comprises a heavy chain variable domain comprising an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to SEQ ID NO:420 and a light chain variabledomain comprising an amino acid sequence at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:421.

In certain embodiments, the present invention provides a proteindisclosed herein comprising a sequence selected from SEQ ID NO:188, SEQID NO:198, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209,SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ IDNO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223,SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:449, SEQ ID NO:450, SEQ IDNO:451, SEQ ID NO:452, SEQ ID NO:453, SEQ ID NO:454, SEQ ID NO:455, SEQID NO:456, SEQ ID NO:457, SEQ ID NO:458, SEQ ID NO:459, SEQ ID NO:460,SEQ ID NO:461, SEQ ID NO:462, SEQ ID NO:463, SEQ ID NO:464, SEQ IDNO:465, SEQ ID NO:466, SEQ ID NO:467, SEQ ID NO:468, SEQ ID NO:469, SEQID NO:470, SEQ ID NO:471, SEQ ID NO:472, SEQ ID NO:473, SEQ ID NO:474,SEQ ID NO:475, SEQ ID NO:476, SEQ ID NO:477, SEQ ID NO:478, SEQ IDNO:479, SEQ ID NO:480, SEQ ID NO:481, SEQ ID NO:482, SEQ ID NO:483, SEQID NO:484, SEQ ID NO:485, SEQ ID NO:486, SEQ ID NO:487, and SEQ IDNO:488.

In certain embodiments, the present invention provides a multi-specificbinding protein disclosed herein comprising an scFv linked to anantibody Fc domain, wherein the scFv linked to the antibody Fc domain isrepresented by a sequence selected from SEQ ID NO:187, SEQ ID NO:197,SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ IDNO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237,SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ IDNO:242, and SEQ ID NO:243.

In certain embodiments, the present invention provides a proteindisclosed herein comprising a sequence selected from SEQ ID NO:189, SEQID NO:196, SEQ ID NO:244, and SEQ ID NO:245.

In certain embodiments, the present invention provides a proteindisclosed herein comprising a sequence at least 90% (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to an aminoacid sequence selected from SEQ ID NO:188, SEQ ID NO:198, SEQ ID NO:206,SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ IDNO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220,SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:447, SEQ IDNO:448, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, SEQID NO:453, SEQ ID NO:454, SEQ ID NO:455, SEQ ID NO:456, SEQ ID NO:457,SEQ ID NO:458, SEQ ID NO:459, SEQ ID NO:460, SEQ ID NO:461, SEQ IDNO:462, SEQ ID NO:463, SEQ ID NO:464, SEQ ID NO:465, SEQ ID NO:466, SEQID NO:467, SEQ ID NO:468, SEQ ID NO:469, SEQ ID NO:470, SEQ ID NO:471,SEQ ID NO:472, SEQ ID NO:473, SEQ ID NO:474, SEQ ID NO:475, SEQ IDNO:476, SEQ ID NO:477, SEQ ID NO:478, SEQ ID NO:479, SEQ ID NO:480, SEQID NO:481, SEQ ID NO:482, SEQ ID NO:483, and SEQ ID NO:484. In certainembodiments, the present invention provides a protein disclosed hereincomprising a sequence at least 95% identical to an amino acid sequenceselected from SEQ ID NO:188, SEQ ID NO:198, SEQ ID NO:206, SEQ IDNO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216,SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ IDNO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:447, SEQ ID NO:448, SEQID NO:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, SEQ ID NO:453,SEQ ID NO:454, SEQ ID NO:455, SEQ ID NO:456, SEQ ID NO:457, SEQ IDNO:458, SEQ ID NO:459, SEQ ID NO:460, SEQ ID NO:461, SEQ ID NO:462, SEQID NO:463, SEQ ID NO:464, SEQ ID NO:465, SEQ ID NO:466, SEQ ID NO:467,SEQ ID NO:468, SEQ ID NO:469, SEQ ID NO:470, SEQ ID NO:471, SEQ IDNO:472, SEQ ID NO:473, SEQ ID NO:474, SEQ ID NO:475, SEQ ID NO:476, SEQID NO:477, SEQ ID NO:478, SEQ ID NO:479, SEQ ID NO:480, SEQ ID NO:481,SEQ ID NO:482, SEQ ID NO:483, and SEQ ID NO:484. In certain embodiments,the present invention provides a protein disclosed herein comprising asequence at least 99% identical to an amino acid sequence selected fromSEQ ID NO:188, SEQ ID NO:198, SEQ ID NO:206, SEQ ID NO:207, SEQ IDNO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217,SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ IDNO:222, SEQ ID NO:223, SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:449, SEQID NO:450, SEQ ID NO:451, SEQ ID NO:452, SEQ ID NO:453, SEQ ID NO:454,SEQ ID NO:455, SEQ ID NO:456, SEQ ID NO:457, SEQ ID NO:458, SEQ IDNO:459, SEQ ID NO:460, SEQ ID NO:461, SEQ ID NO:462, SEQ ID NO:463, SEQID NO:464, SEQ ID NO:465, SEQ ID NO:466, SEQ ID NO:467, SEQ ID NO:468,SEQ ID NO:469, SEQ ID NO:470, SEQ ID NO:471, SEQ ID NO:472, SEQ IDNO:473, SEQ ID NO:474, SEQ ID NO:475, SEQ ID NO:476, SEQ ID NO:477, SEQID NO:478, SEQ ID NO:479, SEQ ID NO:480, SEQ ID NO:481, SEQ ID NO:482,SEQ ID NO:483, and SEQ ID NO:484.

In certain embodiments, the present invention provides a proteindisclosed herein comprising a sequence at least 90% (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to an aminoacid sequence selected from SEQ ID NO:187, SEQ ID NO:197, SEQ ID NO:224,SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ IDNO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238,SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, and SEQ IDNO:243. In certain embodiments, the present invention provides a proteindisclosed herein comprising a sequence at least 95% identical to anamino acid sequence selected from SEQ ID NO:187, SEQ ID NO:197, SEQ IDNO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233,SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ IDNO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, andSEQ ID NO:243. In certain embodiments, the present invention provides aprotein disclosed herein comprising a sequence at least 99% identical toan amino acid sequence selected from SEQ ID NO:187, SEQ ID NO:197, SEQID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228,SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ IDNO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242,and SEQ ID NO:243.

Another aspect of the present invention provides a formulationcomprising a protein as disclosed herein, and a pharmaceuticallyacceptable carrier.

Another aspect of the present invention provides a method of treating aCD33-expressing cancer, the method comprising administering atherapeutically effective amount of a protein or formulation thereofdisclosed herein to a subject in need thereof.

In certain embodiments, the cancer is selected from the group consistingof acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), acutelymphoblastic leukemia (ALL), myeloproliferative neoplasms (MPNs),lymphoma, non-Hodgkin lymphomas, and classical Hodgkin lymphoma. Incertain embodiments, the AML is selected from undifferentiated acutemyeloblastic leukemia, acute myeloblastic leukemia with minimalmaturation, acute myeloblastic leukemia with maturation, acutepromyelocytic leukemia (APL), acute myelomonocytic leukemia, acutemyelomonocytic leukemia with eosinophilia, acute monocytic leukemia,acute erythroid leukemia, acute megakaryoblastic leukemia (AMKL), acutebasophilic leukemia, acute panmyelosis with fibrosis, and blasticplasmacytoid dendritic cell neoplasm (BPDCN). In certain embodiments,the AML is characterized by expression of CLL-1 on the AML leukemia stemcells (LSCs). In certain embodiments, the LSCs further express amembrane marker selected from CD34, CD38, CD123, TIM3, CD25, CD32, andCD96.

In certain embodiments, the AML is a minimal residual disease (MRD). Incertain embodiments, the MRD is characterized by the presence or absenceof a mutation selected from FLT3-ITD ((Fms-like tyrosine kinase3)-internal tandem duplications (ITD)), NPM1 (Nucleophosmin 1), DNMT3A(DNA methyltransferase gene DNMT3A), and IDH (Isocitrate dehydrogenase 1and 2 (IDH1 and IDH2)). In certain embodiments, the MDS is selected fromMDS with multilineage dysplasia (MDS-MLD), MDS with single lineagedysplasia (MDS-SLD), MDS with ring sideroblasts (MDS-RS), MDS withexcess blasts (MDS-EB), MDS with isolated del(5q), and MDS, unclassified(MDS-U). In certain embodiments, the MDS is a primary MDS or a secondaryMDS.

In certain embodiments, the ALL is selected from B-cell acutelymphoblastic leukemia (B-ALL) and T-cell acute lymphoblastic leukemia(T-ALL). In certain embodiments, the MPN is selected from polycythaemiavera, essential thrombocythemia (ET), and myelofibrosis. In certainembodiments, the non-Hodgkin lymphoma is selected from B-cell lymphomaand T-cell lymphoma. In certain embodiments, the lymphoma is selectedfrom chronic lymphocytic leukemia (CLL), lymphoblastic lymphoma (LPL),diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), primarymediastinal large B-cell lymphoma (PMBL), follicular lymphoma, mantlecell lymphoma, hairy cell leukemia, plasma cell myeloma (PCM) ormultiple myeloma (MM), mature T/NK neoplasms, and histiocytic neoplasms.

These and other aspects and advantages of the invention are illustratedby the following figures, detailed description and claims.

DESCRIPTION OF THE DRAWINGS

The invention can be more completely understood with reference to thefollowing drawings.

FIG. 1 shows a structural representation of the extracellular domain ofhuman CD33 extracellular domain (ECD). CD33 ECD contains two prominentdomains: distal V domain and membrane proximal C domain. Ligand bindinginterface is located on the V domain. Function of the C domain isunknown. ECD of CD33 is heavily glycosylated, with 2 N-linkedglycosylation sites located in the V domain and 3 N-linked glycosylationsites located in the C domain. ECD of human CD33 contains several SNPs,with most prominent mutation R69G that is found in 42% patients. SNPR69G is in the V domain.

FIG. 2 shows an alignment of the primary sequences of full length humanand cyno CD33 (SEQ ID NO:598 and SEQ ID NO:599, respectively). V domainis underlined in blue, C domain is underlined in green. Difference insequences are framed in red.

FIGS. 3A-3K show SPR profiles of Fab fragments from CD33 monoclonalantibodies binding to human CD33 ECD measured by Biacore at 37° C. EachFab fragment includes a CD33-binding clone described herein. FIG. 3A isa Biacore profile of ADI-10159; FIG. 3B is a Biacore profile ofADI-10177; FIG. 3C is a Biacore profile of ADI-11776; FIG. 3D is aBiacore profile of ADI-11801; FIG. 3E is a Biacore profile of ADI-11807;FIG. 3F is a Biacore profile of ADI-11809; FIG. 3G is a Biacore profileof ADI-11815; FIG. 3H is a Biacore profile of ADI-11819; FIG. 3I is aBiacore profile of ADI-11830; FIG. 3J is a Biacore profile of ADI-11835;and FIG. 3K is a Biacore profile of Fab fragment from Lintuzumab.

FIGS. 4A-4H show SPR profiles of Fab fragments from CD33 monoclonalantibodies binding to cyno CD33 ECD measured by Biacore at 37° C. EachFab fragment includes a CD33-binding clone described herein. FIG. 4A isa Biacore profile of ADI-10159; FIG. 4B is a Biacore profile ofADI-10177; FIG. 4C is a Biacore profile of ADI-11776; FIG. 4D is aBiacore profile of ADI-11807; FIG. 4E is a Biacore profile of ADI-11809;FIG. 4F is a Biacore profile of ADI-11819; FIG. 4G is a Biacore profileof ADI-11830; and FIG. 4H is a Biacore profile of ADI-11835.

FIGS. 5A-5T show SPR profiles of FABs from CD33 monoclonal antibodiesbinding to V domain and C domain of human CD33 measured at 37° C. EachFab fragment includes a CD33-binding clone described herein. FIGS. 5A-5Jrepresent binding to the V-domain; FIGS. 5K-5T represent binding to theC domain. FIGS. 5A and 5K are Biacore profiles of ADI-10159; FIGS. 5Band 5L are Biacore profiles of ADI-10177; FIGS. 5C and 5M are Biacoreprofiles of ADI-11776; FIGS. 5D and 5N are Biacore profiles ofADI-11801; FIGS. 5E and 5O are Biacore profiles of ADI-11807; FIGS. 5Fand 5P are Biacore profiles of ADI-11809; FIGS. 5G and 5Q are Biacoreprofiles of ADI-11815; FIGS. 5H and 5R are Biacore profiles ofADI-11819; FIGS. 5I and 5S are Biacore profiles of ADI-11830; and FIGS.5J and 5T are Biacore profiles of ADI-11835.

FIGS. 6A-6D show SPR profiles of an Fab that comprises ADI-11815 bindingto different domains of human CD33 and human CD33 having an R69G pointmutation. FIG. 6A: Fab binding to human CD33 ECD; FIG. 6B: Fab bindingto V domain; FIG. 6C: Fab binding to C domain: FIG. 6D: Fab binding tohuman CD33 having R69G.

FIGS. 7A-7D show SPR profiles of a Fab that comprises ADI-11801 bindingto different domains of human CD33 and human CD33 having an R69G pointmutation. FIG. 7A: human CD33 ECD; FIG. 7B: V domain; FIG. 7C: C domain:FIG. 7D: human CD33 having R69G.

FIG. 8 are bar graphs showing binding of monoclonal antibodiescomprising CD33-binding clones to CD33 expressed on Molm-13 human AMLcells. CD33 antibody Lintuzumab was also tested, and mean fluorescenceintensity (MFI) was plotted. Five of the six antibodies show higherbinding signal to CD33 compared to Lintuzumab.

FIG. 9 are bar graphs showing internalization of CD33 antibodies onMolm-13 cells after 24 hours. All the CD33 antibodies showed similarinternalization after 24 hours. Lintuzumab showed slightly higherinternalization compare to other anti-CD33 antibodies.

FIGS. 10A-10B show binding of CD33-targeting TriNKETs to human NKG2Dexpressed on EL4-hNKG2D and KHYG-1 cells. FIG. 10A shows binding ofCD33-targeting TriNKETs to human NKG2D recombinantly expressed on EL4cells. FIG. 10B shows binding of CD33-targeting TriNKETs to human NKG2Dexpressed on KHYG-1 cells. For each TriNKET, signal fold-over-background(FOB) was similar on both EL4-hNKG2D cells and KHYG-1 cells, and therank of binding was also maintain on both cell lines.

FIG. 11 shows binding of CD33-targeting TriNKETs to CD33 expressed onhuman AML Molm-13 cells. Four different CD33-binding clones were usedwith five NKG2D-binding clones to make a total of 20 different TriNKETs.NKG2D-binding domains TriNKET do not affect the binding of CD33-bindingclones to CD33.

FIG. 12 is a graph showing that rested human NK cells are activated byCD33-targeting TriNKETs in co-culture with CD33-expressing THP-1 AMLcells.

FIG. 13 is a bar graph showing that CD33 TriNKETs induce rested NK cellmediated killing of Molm-13 AML cells.

FIG. 14 is a bar graph showing that CD33 TriNKETs induce activated NKcell mediated killing of THP-1 cells.

FIG. 15A are line graphs showing that TriNKETs mediate KHYG-1 killing ofMolm-13 AML cells. FIG. 15B are line graphs showing that TriNKETsmediate rested human NK cell killing of Molm-13 human AML cells.

FIG. 16 are line graphs showing that TriNKETs mediate KHYG-1 killing ofEOL-1 AML cells.

FIG. 17A are line graphs showing that TriNKETs mediate KHYG-1 killing ofTHP-1 cells. FIG. 17B are line graphs showing that TriNKETs mediaterested human NK cell killing of THP-1 human AML cells.

FIG. 18 is a representation of a multispecific binding protein thatcontains an NKG2D-binding domain (right arm), a CD33-binding domain(left arm), and an Fc domain or a portion thereof that binds to CD16.

FIG. 19 is a representation of a multispecific binding protein thatincludes a NKG2D-binding domain or a CD33-binding domain, either one ofwhich can be in a scFv format, and an Fc domain or a portion thereofthat binds to CD16.

FIG. 20 is a representation of a TriNKET in the Triomab form, which is atrifunctional, bispecific antibody that maintains an IgG-like shape.This chimera consists of two half antibodies, each with one light andone heavy chain, that originate from two parental antibodies.

FIG. 21 is a representation of a TriNKET in the KiH Common Light Chain(LC) form, which involves the knobs-into-holes (KIHs) technology. KiH isa heterodimer containing 2 Fabs binding to target 1 and 2, and an Fcstabilized by heterodimerization mutations. TriNKET in the KiH formatmay be an heterodimeric construct with 2 Fabs binding to target 1 andtarget 2, containing two different heavy chains and a common light chainthat pairs with both heavy chains.

FIG. 22 is a representation of a TriNKET in the dual-variable domainimmunoglobulin (DVD-Ig™) form, which combines the target binding domainsof two monoclonal antibodies via flexible naturally occurring linkers,and yields a tetravalent IgG-like molecule. DVD-Ig™ is a homodimericconstruct where variable domain targeting antigen 2 is fused to the Nterminus of variable domain of Fab targeting antigen 1 Constructcontains normal Fc.

FIG. 23 is a representation of a TriNKET in the Orthogonal Fab interface(Ortho-Fab) form, which is an heterodimeric construct that contains 2Fabs binding to target 1 and target 2 fused to Fc. LC-HC pairing isensured by orthogonal interface. Heterodimerization is ensured bymutations in the Fc.

FIG. 24 is a representation of a TrinKET in the 2-in-1 Ig format.

FIG. 25 is a representation of a TriNKET in the ES form, which is anheterodimeric construct containing two different Fabs binding to target1 and target 2 fused to the Fc. Heterodimerization is ensured byelectrostatic steering mutations in the Fc.

FIG. 26 is a representation of a TriNKET in the Fab Arm Exchange form:antibodies that exchange Fab arms by swapping a heavy chain and attachedlight chain (half-molecule) with a heavy-light chain pair from anothermolecule, resulting in bispecific antibodies. Fab Arm Exchange form(cFae) is a heterodimer containing 2 Fabs binding to target 1 and 2, andan Fc stabilized by heterodimerization mutations.

FIG. 27 is a representation of a TriNKET in the SEED Body form, which isan heterodimer containing two Fabs binding to target 1 and 2, and an Fcstabilized by heterodimerization mutations.

FIG. 28 is a representation of a TriNKET in the LuZ-Y form, in whichleucine zipper is used to induce heterodimerization of two differentHCs. LuZ-Y form is a heterodimer containing two different scFabs bindingto target 1 and 2, fused to Fc.

FIG. 29 is a representation of a TriNKET in the Cov-X-Body form.

FIGS. 30A-30B represent TriNKETs in the κλ-Body forms, which are anheterodimeric constructs with two different Fabs fused to Fc stabilizedby heterodimerization mutations: Fab 1 targeting antigen 1 containskappa LC, while second Fab targeting antigen 2 contains lambda LC. FIG.30A is an exemplary representation of one form of a κλ-Body;

FIG. 30B is an exemplary representation of another κλ-Body.

FIG. 31 is an Oasc-Fab heterodimeric construct that includes Fab bindingto target 1 and scFab binding to target 2 fused to Fc.Heterodimerization is ensured by mutations in the Fc.

FIG. 32 is a DuetMab, which is an heterodimeric construct containing twodifferent Fabs binding to antigens 1 and 2, and Fc stabilized byheterodimerization mutations. Fab 1 and 2 contain differential S-Sbridges that ensure correct light chain (LC) and heavy chain (HC)pairing.

FIG. 33 is a CrossmAb, which is an heterodimeric construct with twodifferent Fabs binding to targets 1 and 2 fused to Fc stabilized byheterodimerization. CL and CH1 domains and VH and VL domains areswitched, e.g., CH1 is fused in-line with VL, while CL is fused in-linewith VH.

FIG. 34 is a Fit-Ig, which is a homodimeric constructs where Fab bindingto antigen 2 is fused to the N terminus of HC of Fab that binds toantigen 1. The construct contains wild-type Fc.

FIG. 35 is a graph showing binding of A49-F3′-TriNKET-I07 and I07-F405LmAb to cell surface human NKG2D expressed on EL4 cells.

FIGS. 36A-36B are graphs showing binding of A49-F3′-TriNKET-I07 andI07-F405L mAb to CD33⁺ human AML cell lines Mv4-11 (FIG. 36A) andMolm-13 (FIG. 36B).

FIGS. 37A-37B are graphs showing internalization of A49-F3′-TriNKET-I07and I07-F405L mAb after incubation with EOL-1 cells (FIG. 37A) andMolm-13 cells (FIG. 37B).

FIGS. 38A-38D are graphs showing specific lysis of Molm-13 (FIG. 38A),EOL-1 (FIG. 38B), and THP-1 (FIGS. 38C and 38D) human AML cells byrested human NK cells in the presence of A49-F3′-TriNKET-I07 andanti-CD33 monoclonal antibodies.

FIG. 39 is a series of flow cytograms showing the expression level ofCD3, CD8, NKG2D, and CD16 on isolated primary CD8⁺ T cells.

FIGS. 40A-40B are graphs showing specific lysis of Molm-13 cells byisolated primary CD8⁺ T cells in the presence of A49-F3′-TriNKET-I07,A49-F3′-TriNKET-H76, a non-target TriNKET, or I07-F405L mAb (denoted asI07 in the figures). The primary CD8⁺ T cells in FIG. 40A were isolatedfrom PBMCs of donor 1, and the primary CD8⁺ T cells in FIG. 40B wereisolated from PBMCs of donor 2. The dotted lines indicate specific lysisof Molm-13 cells by CD8⁺ T cells in the absence of TriNKET or antibody.

FIGS. 41A-41E are histograms showing the binding of A49-F3′-TriNKET-I07to NK cells (FIG. 41A), CD8⁺ T cells (FIG. 41B), CD4⁺ T cells (FIG.41C), B cells (FIG. 41D), and monocytes (FIG. 41E) in human whole blood.The dotted lines without fill represent binding of A49-F3′-TriNKET-I07to the cells; the solid lines with fill represent binding of human IgG1isotype control to the cells.

FIGS. 42A-42B are graphs showing CD33 expression on monocytes. FIG. 42Ashows CD33 expression on monocytes from four healthy donors (dark gray)and Molm-13 (light grey). The bottom five rows are signals from the cellsamples stained with an anti-CD33 antibody; the top five rows aresignals from the same samples stained with an isotype antibody. FIG. 42Bshows CD33 expression on monocytes from the same donor before (lightgrey) and after (dark grey) negative selection for monocytes.

FIGS. 43A-43B are graphs showing long-term cytotoxicity of NK cellsagainst Molm-13 AML cells and human primary monocytes in the presence ofA49-F3′-TriNKET-I07. Proliferation of the target cells are plottedagainst the time of co-culture with NK cells in the presence ofA49-F3′-TriNKET-I07 or PMA+ionomycin. FIG. 43A represents the resultsfrom an experiment using NK cells from one donor, and FIG. 43Brepresents the results from another experiment using NK cells from adifferent donor.

FIG. 44 illustrates a trispecific antibody (TriNKET) that contains aCD33-binding scFv, a NKG2D-targeting Fab, and a heterodimerized antibodyconstant domain that binds CD16. The antibody format is referred toherein as F3′-TriNKET.

DETAILED DESCRIPTION

The invention provides multi-specific binding proteins that bind a CD33on a cancer cell and the NKG2D receptor and CD16 receptor on naturalkiller cells to activate the natural killer cell, pharmaceuticalcompositions comprising such multi-specific binding proteins, andtherapeutic methods using such multi-specific proteins andpharmaceutical compositions, including for the treatment of cancer.Various aspects of the invention are set forth below in sections;however, aspects of the invention described in one particular sectionare not to be limited to any particular section.

In one aspect, the invention provides a multi-specific binding proteincomprising an antigen binding site that binds an epitope on anextracellular domain of human CD33 and/or Cynomolgus/Rhesus (cyno) CD33.In one aspect, the present invention provides an antigen binding sitethat recognizes and binds one or more conformational epitopes on theextracellular domain of human CD33 and/or Cynomolgus/Rhesus (cyno) CD33.In one aspect, the present invention provides an antigen binding sitethat recognizes and binds one or more conformational epitopes on theextracellular domain of human CD33 but does not recognize and/or bindone or more conformational epitopes on the extracellular domain of cynoCD33. In one aspect, the present invention provides an antigen bindingsite that binds to the R69G allele of human CD33. In one aspect, thepresent invention provides an antigen binding site that binds towild-type human CD33 but not the R69G allele of human CD33. In oneaspect, the present invention provides an antigen binding site thatbinds to an epitope on human CD33 that includes R69. In one aspect, thepresent invention provides an antigen binding site that binds to theS128N allele of human CD33. In one aspect, the present inventionprovides an antigen binding site that binds to wild-type human CD33 butnot the S128N allele of human CD33. In one aspect, the present inventionprovides an antigen binding site that binds to an epitope on human CD33that includes S128. In one aspect, the present invention provides anantigen binding site including a heavy chain variable domain, whichbinds to the extracellular domain in human CD33 and/or cyno CD33,irrespective of the glycosylation profile of the targeted CD33.

In certain embodiments the present invention provides a multi-specificbinding protein comprising an antigen binding site, which binds to theextracellular domain in human CD33 and/or cyno CD33, such that theepitopes are unique compared to the epitopes targeted by one or moreknown anti-CD33 antibodies in the art. In certain embodiments, thepresent invention provides an antigen binding site, which binds to theextracellular domain in human CD33 and/or cyno CD33, and shows human orCynomolgus/Rhesus (cyno) CD33 cross reactivity and high affinity bindingto the target CD33.

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The terms “a” and “an” as used herein mean “one or more” and include theplural unless the context is inappropriate.

As used herein, the term “antigen-binding site” refers to the part ofthe immunoglobulin molecule that participates in antigen binding. Inhuman antibodies, the antigen binding site is formed by amino acidresidues of the N-terminal variable (“V”) regions of the heavy (“H”) andlight (“L”) chains. Three highly divergent stretches within the Vregions of the heavy and light chains are referred to as “hypervariableregions” which are interposed between more conserved flanking stretchesknown as “framework regions,” or “FR.” Thus the term “FR” refers toamino acid sequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In a human antibody molecule,the three hypervariable regions of a light chain and the threehypervariable regions of a heavy chain are disposed relative to eachother in three dimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.” In certain animals,such as camels and cartilaginous fish, the antigen-binding site isformed by a single antibody chain providing a “single domain antibody.”Antigen-binding sites can exist in an intact antibody, in anantigen-binding fragment of an antibody that retains the antigen-bindingsurface, or in a recombinant polypeptide such as an scFv, using apeptide linker to connect the heavy chain variable domain to the lightchain variable domain in a single polypeptide. All the amino acidpositions in heavy or light chain variable regions disclosed herein arenumbered according to Kabat numbering.

The CDRs of an antigen-binding site can be determined by the methodsdescribed in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) andKabat et al., Sequences of protein of immunological interest. (1991),Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al.,J. Mol. Biol. 262:732-745 (1996). The CDRs determined under thesedefinitions typically include overlapping or subsets of amino acidresidues when compared against each other. In certain embodiments, theterm “CDR” is a CDR as defined by MacCallum et al., J. Mol. Biol.262:732-745 (1996) and Martin A., Protein Sequence and StructureAnalysis of Antibody Variable Domains, in Antibody Engineering,Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag,Berlin (2001). In certain embodiments, the term “CDR” is a CDR asdefined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabatet al., Sequences of protein of immunological interest. (1991). Incertain embodiments, heavy chain CDRs and light chain CDRs of anantibody are defined using different conventions. For example, incertain embodiments, the heavy chain CDRs are defined according toMacCallum (supra), and the light CDRs are defined according to Kabat(supra). CDRH1, CDRH2 and CDRH3 denote the heavy chain CDRs, and CDRL1,CDRL2 and CDRL3 denote the light chain CDRs.

As used herein, the terms “subject” and “patient” refer to an organismto be treated by the methods and compositions described herein. Suchorganisms preferably include, but are not limited to, mammals (e.g.,murines, simians, equines, bovines, porcines, canines, felines, and thelike), and more preferably include humans.

As used herein, the term “effective amount” refers to the amount of acompound (e.g., a compound of the present invention) sufficient toeffect beneficial or desired results. An effective amount can beadministered in one or more administrations, applications or dosages andis not intended to be limited to a particular formulation oradministration route. As used herein, the term “treating” includes anyeffect, e.g., lessening, reducing, modulating, ameliorating oreliminating, that results in the improvement of the condition, disease,disorder, and the like, or ameliorating a symptom thereof.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent with a carrier, inert or active, makingthe composition especially suitable for diagnostic or therapeutic use invivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions (e.g., such as an oil/wateror water/oil emulsions), and various types of wetting agents. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. [1975].

As used herein, the term “pharmaceutically acceptable salt” refers toany pharmaceutically acceptable salt (e.g., acid or base) of a compoundof the present invention which, upon administration to a subject, iscapable of providing a compound of this invention or an activemetabolite or residue thereof. As is known to those of skill in the art,“salts” of the compounds of the present invention may be derived frominorganic or organic acids and bases. Exemplary acids include, but arenot limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic,toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,benzenesulfonic acid, and the like. Other acids, such as oxalic, whilenot in themselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts.

Exemplary bases include, but are not limited to, alkali metal (e.g.,sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Exemplary salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.Other examples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like.

For therapeutic use, salts of the compounds of the present invention arecontemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable compound.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes andmethods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions ofthe present invention that consist essentially of, or consist of, therecited components, and that there are processes and methods accordingto the present invention that consist essentially of, or consist of, therecited processing steps.

As a general matter, compositions specifying a percentage are by weightunless otherwise specified. Further, if a variable is not accompanied bya definition, then the previous definition of the variable controls.

Various features and aspects of the invention are discussed in moredetail below.

I. Antigen-Binding Site

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:1, and an antibody light chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQID NO:2. In certain embodiments, an antigen-binding site that includesan antibody heavy chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:1, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:21, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:22,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:23. In certain embodiments, an antigen-binding site that includes anantibody heavy chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:1, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:434, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:22,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:435. In certain embodiments, an antigen-binding site that includes anantibody light chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:2, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:24, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:25,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:26.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:3, and an antibody light chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQID NO:4. In certain embodiments, an antigen-binding site that includesan antibody heavy chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:3, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:27, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:28,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:29. In certain embodiments, an antigen-binding site that includes anantibody heavy chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:3, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:181, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:28,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:436. In certain embodiments, an antigen-binding site that includes anantibody light chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:4, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:30, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:31,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:32.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:5, and an antibody light chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQID NO:6. In certain embodiments, an antigen-binding site that includesan antibody heavy chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:5, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:33, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:34,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:35. In certain embodiments, an antigen-binding site that includes anantibody heavy chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:5, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:183, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:34,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:184. In certain embodiments, an antigen-binding site that includes anantibody light chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:6, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:36, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:37,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:38. In certain embodiments, an antigen-binding site that includes anantibody light chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:6, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:36, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:185,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:38. In certain embodiments, the antigen-binding site comprises anamino acid sequence at least 90% (e.g., at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:188. Incertain embodiments, the antigen-binding site comprises an amino acidsequence at least 95% identical to SEQ ID NO:188. In certainembodiments, the antigen-binding site comprises an amino acid sequenceat least 99% identical to SEQ ID NO:188. In certain embodiments, theantigen-binding site comprises the amino acid sequence of SEQ ID NO:188.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:7, and an antibody light chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQID NO:8. In certain embodiments, an antigen-binding site that includesan antibody heavy chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:7, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:39, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:40,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:41. In certain embodiments, an antigen-binding site that includes anantibody heavy chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:7, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:437, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:40,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:438. In certain embodiments, an antigen-binding site that includes anantibody light chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:8, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:42, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:43,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:44.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:9, and an antibody light chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQID NO:10. In certain embodiments, an antigen-binding site that includesan antibody heavy chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:9, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:45, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:46,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:47. In certain embodiments, an antigen-binding site that includes anantibody heavy chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:9, includes aCDR1 sequence represented by the amino acid sequence of SEQ ID NO:181, aCDR2 sequence represented by the amino acid sequence of SEQ ID NO:46,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:182. In certain embodiments, an antigen-binding site that includes anantibody light chain variable domain with an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:10, includesa CDR1 sequence represented by the amino acid sequence of SEQ ID NO:48,a CDR2 sequence represented by the amino acid sequence of SEQ ID NO:49,and a CDR3 sequence represented by the amino acid sequence of SEQ IDNO:50. In certain embodiments, the antigen-binding site comprises anamino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:198. In certainembodiments, the antigen-binding site comprises an amino acid sequenceat least 95% identical to SEQ ID NO:198. In certain embodiments, theantigen-binding site comprises an amino acid sequence at least 99%identical to SEQ ID NO:198. In certain embodiments, the antigen-bindingsite comprises the amino acid sequence of SEQ ID NO:198.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:11, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:12. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:51, a CDR2 sequence represented by the amino acid sequence of SEQID NO:52, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:53. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:181, a CDR2 sequence represented by the amino acid sequence of SEQID NO:52, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:439. In certain embodiments, an antigen-binding site thatincludes an antibody light chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:12,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:54, a CDR2 sequence represented by the amino acid sequence of SEQID NO:55, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:56.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:13, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:14. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:13,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:57, a CDR2 sequence represented by the amino acid sequence of SEQID NO:58, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:59. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:13,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:440, a CDR2 sequence represented by the amino acid sequence of SEQID NO:58, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:441. In certain embodiments, an antigen-binding site thatincludes an antibody light chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:14,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:60, a CDR2 sequence represented by the amino acid sequence of SEQID NO:61, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:62.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:15, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:16. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:15,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:63, a CDR2 sequence represented by the amino acid sequence of SEQID NO:64, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:65. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:15,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:442, a CDR2 sequence represented by the amino acid sequence of SEQID NO:64, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:443. In certain embodiments, an antigen-binding site thatincludes an antibody light chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:16,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:66, a CDR2 sequence represented by the amino acid sequence of SEQID NO:67, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:68.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:17, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:18. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:17,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:69, a CDR2 sequence represented by the amino acid sequence of SEQID NO:70, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:71. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:17,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:181, a CDR2 sequence represented by the amino acid sequence of SEQID NO:70, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:444. In certain embodiments, an antigen-binding site thatincludes an antibody light chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:18,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:72, a CDR2 sequence represented by the amino acid sequence of SEQID NO:73, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:74.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:19, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:20. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:19,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:75, a CDR2 sequence represented by the amino acid sequence of SEQID NO:76, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:77. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:19,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:445, a CDR2 sequence represented by the amino acid sequence of SEQID NO:76, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:446. In certain embodiments, an antigen-binding site thatincludes an antibody light chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:20,includes a CDR1 sequence represented by the amino acid sequence of SEQID NO:78, a CDR2 sequence represented by the amino acid sequence of SEQID NO:79, and a CDR3 sequence represented by the amino acid sequence ofSEQ ID NO:80.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:266, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:267. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:266, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:304, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:305, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:306. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:266, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:528, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:305, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:529. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:267, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:307, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:308, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:309.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:268, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:269. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:268, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:310, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:311, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:312. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:268, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:530, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:311, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:531. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:269, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:313, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:314, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:315.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:270, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:271. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:270, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:316, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:317, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:318. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:270, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:532, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:317, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:533. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:271, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:319, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:320, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:321.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:272, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:273. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:272, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:322, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:323, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:324. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:272, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:534, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:323, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:535. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:273, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:325, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:326, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:327.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:274, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:275. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:274, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:328, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:329, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:330. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:274, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:536, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:329, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:537. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:275, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:331, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:332, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:333.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:276, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:277. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:276, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:334, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:335, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:336. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:276, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:538, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:335, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:539. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:277, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:337, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:338, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:339.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:278, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:279. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:278, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:340, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:341, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:342. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:278, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:540, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:341, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:541. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:279, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:343, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:344, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:345.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:280, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:281. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:280, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:346, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:347, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:348. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:280, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:542, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:347, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:543. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:281, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:349, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:350, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:351.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:282, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:283. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:282, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:352, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:353, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:354. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:282, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:544, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:353, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:545. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:283, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:355, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:356, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:357.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:284, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:285. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:284, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:358, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:359, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:360. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:284, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:546, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:359, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:547. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:285, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:361, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:362, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:363.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:286, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:287. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:286, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:364, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:365, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:366. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:286, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:548, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:365, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:549. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:287, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:367, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:368, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:369.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:288, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:289. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:288, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:370, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:371, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:372. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:288, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:550, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:371, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:551. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:289, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:373, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:374, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:375.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:290, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:291. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:290, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:376, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:377, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:378. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:290, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:552, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:377, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:553. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:291, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:379, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:380, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:381.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:292, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:293. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:292, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:382, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:383, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:384. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:292, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:554, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:383, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:555. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:293, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:385, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:386, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:387.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:294, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:295. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:294, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:388, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:389, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:390. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:294, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:556, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:389, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:557. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:295, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:391, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:392, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:393.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:296, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:297. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:296, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:394, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:395, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:396. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:296, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:558, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:395, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:559. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:297, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:397, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:398, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:399.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:298, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:299. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:298, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:400, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:401, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:402. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:298, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:560, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:401, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:561. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:299, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:403, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:404, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:405.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:300, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:301. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:300, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:406, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:407, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:408. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:300, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:562, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:407, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:563. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:301, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:409, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:410, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:411.

In certain embodiments, the present invention provides anantigen-binding site that includes an antibody heavy chain variabledomain that includes an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:302, and an antibody light chainvariable domain that includes an amino acid sequence at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto SEQ ID NO:303. In certain embodiments, an antigen-binding site thatincludes an antibody heavy chain variable domain with an amino acidsequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:302, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:412, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:413, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:414. In certain embodiments, an antigen-bindingsite that includes an antibody heavy chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:302, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:564, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:413, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:565. In certain embodiments, an antigen-bindingsite that includes an antibody light chain variable domain with an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:303, includes a CDR1 sequence represented by the amino acid sequenceof SEQ ID NO:415, a CDR2 sequence represented by the amino acid sequenceof SEQ ID NO:416, and a CDR3 sequence represented by the amino acidsequence of SEQ ID NO:417.

In each of the foregoing embodiments, it is contemplated herein thatimmunoglobulin heavy chain variable region sequences and/or light chainvariable region sequences that together bind CD33 may contain amino acidalterations (e.g., at least 1, 2, 3, 4, 5, or 10 amino acidsubstitutions, deletions, or additions) in the framework regions of theheavy and/or light chain variable regions without affecting theirability to bind to CD33 significantly.

Table 1 lists peptide sequences of heavy chain variable domains andlight chain variable domains that, in combination (either as a Fabfragment or a single-chain variable fragment (scFv)), can bind to CD33.Unless indicated otherwise, the CDR sequences provided in Table 1 aredetermined under Kabat. The CD33-binding domains can vary in theirbinding affinity to CD33. Table 1 also lists scFv forms of theCD33-binding heavy and light chain variable domains. The exemplarynucleic acid sequences listed in Table 1 are predicted possible nucleicacid sequences that the listed corresponding peptide sequencesoriginated from, and were generated using EMBL-EBI's Protein SequenceBack-translation program.

TABLE 1 VH VL ADI-10159 EVQLVESGGGLVQPGGSLRLSC DIQMTQSPSTLSASVGDRVTITCR[Ab1] AASGFTFSSYGMSWVRQAPGK ASQSISSWLAWYQQKPGKAPKL (G59)GLEWVANIKQDGSEKYYVDSV LIYDASSLESGVPSRFSGSGSGTE KGRFTISRDNAKNSLYLQMNSLFTLTISSLQPDDFATYYCQQYESF RAEDTAVYYCAREGGPYYDSSPTFGGGTKVEIK [SEQ ID NO: 2] GYFVYYGMDVWGQGTTVTVSSCDR1: RASQSISSWLA [SEQ ID [SEQ ID NO: 1] NO: 24] CDR1: FTFSSYGMS [SEQ IDCDR2: DASSLES [SEQ ID NO: 25] NO: 21](non-Kabat) or SYGMSCDR3: QQYESFPT [SEQ ID NO: 26] [SEQ ID NO: 434] CDR2: NIKQDGSEKYYVDSVKG[SEQ ID NO: 22] CDR3: AREGGPYYDSSGYFVYYGMDV[SEQ ID NO: 23](non-Kabat) or EGGPYYDSSGYFVYYGMDV [SEQ ID NO: 435]scFv of DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLI Ab1YDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESFPTF G

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQVESGGGLVQPG GSLRLSCAASGFTFSSYGMSWVRQAPGK

LEWVANIKQDGSEKYYV DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS [SEQ ID NO: 206]EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGK

LE WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS GGGGSGGG GSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDD FATYYCQQYESFPTFG

GTKVEIK [SEQ ID NO: 207] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCA sequenceGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab1AAGCTGCTGATCTACGACGCCAGCAGCCTGGAGAGCGGCGTGCC scFvCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACGAGAGCTTCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGGCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCCCCTACTACGACAGCAGCGGCTACTTCGTGTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGAC CGTGAGCAGC [SEQ ID NO: 246]GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGGCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCCCCTACTACGACAGCAGCGGCTACTTCGTGTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACGAGAGCTTCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 247] ADI-10177 EVQLVESGGGLVQPGGSLRLSCDIQMTQSPSTLSASVGDRVTITCR [Ab2] AASGFTFSSYWMSWVRQAPGKASQSISSWLAWYQQKPGKAPKL GLEWVANIKQDGSEKYYVDSV LIYEASSLESGVPSRFSGSGSGTEKGRFTISRDNAKNSLYLQMNSL FTLTISSLQPDDFATYYCQQLESY RAEDTAVYYCARPLNAGELDVPLTFGGGTKVEIK [SEQ ID NO: 4] WGQGTMVTVSS [SEQ ID NOG]CDR1: RASQSISSWLA [SEQ ID CDR1: FTFSSYWMS [SEQ ID NO: 30]NO: 27](non-Kabat) or SYWMS CDR2: EASSLES [SEQ ID NOG 1][SEQ ID NO: 181] CDR3: QQLESYPLT [SEQ ID CDR2: NIKQDGSEKYYVDSVKG NOG2][SEQ ID NO: 28] CDR3: ARPLNAGELDV [SEQ ID NO: 29](non-Kabat) orPLNAGELDV [SEQ ID NO: 436] scFv ofDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLI Ab2YEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLESYPLT FG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LEWVANIKQDGSEKY YVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS [SEQ ID NO: 208]EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LE WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS GGGGSGGGGSGGGGSGG GGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLES YPLTFG

GTKVEIK [SEQ ID NO: 209] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCA sequenceGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab2AAGCTGCTGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCC scFvCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGCTGGAGAGCTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCATGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCCCCTGAACGCCGGCGAGCTGGACGTGTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC [SEQ ID NO: 248]GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCCCCTGAACGCCGGCGAGCTGGACGTGTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGCTGGAGAGCTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 249] ADI-11776EVQLLESGGGLVQPGGSLRLSC DIQMTQSPSTLSASVGDRVTITCR [Ab3]AASGFTFSKYTMSWVRQAPGK ASQSISSWLAWYQQKPGKAPKL (H76)GLEWVSAIVGSGESTYFADSVK LIYKASSLESGVPSRFSGSGSGTE GRFTISRDNSKNTLYLQMNSLRFTLTISSLQPDDFATYYCQQYDD AEDTAVYYCAREGGPYYDSSGLPTFGGGTKVEIK [SEQ ID NO: 6] YFVYYGMDVWGQGTTVTVSSCDR1: RASQSISSWLA [SEQ ID [SEQ ID NO: 5] NO: 36] CDR1: FTFSKYTMS [SEQ IDCDR2: KASSLES [SEQ ID NO: 37] NO: 33](non-Kabat) or KYTMSor KASSLE [SEQ ID NO: 185](non- [SEQ ID NO: 183] Kabat)CDR2: AIVGSGESTYFADSVKG CDR3: QQYDDLPT [SEQ ID [SEQ ID NO: 34] NO: 38]CDR3: AREGGPYYDSSGYFVYYGMDV [SEQ ID NO: 35](non-Kabat) orEGGPYYDSSGYFVYYGMDV [SEQ ID NO: 184] scFv ofDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLI Ab3YKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPT FG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMSWVRQAPGK

LEWVSAIVGSGESTYF ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS [SEQ ID NO: 198]EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMSWVRQAPGK

LE WVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS GGGGSGGGG SGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFA TYYCQQYDDLPTFG

GTKVEIK [SEQ ID NO: 210] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCA sequenceGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab 3AAGCTGCTGATCTACAAGGCCAGCAGCCTGGAGAGCGGCGTGCC scFvCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACGACGACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAAGTACACCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGAGCGCCATCGTGGGCAGCGGCGAGAGCACCTACTTCGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCCCCTACTACGACAGCAGCGGCTACTTCGTGTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGAC CGTGAGCAGC [SEQ ID NO: 250]GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAAGTACACCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGAGCGCCATCGTGGGCAGCGGCGAGAGCACCTACTTCGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCCCCTACTACGACAGCAGCGGCTACTTCGTGTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACGACGACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 251] ADI-11801 QVQLVQSGAEVKKPGASVKVSDIVMTQSPLSLPVTPGEPASISCR [Ab4] CKASGYTFSDYYMHWVRQAPGSSQSLLYSNGYNYLDWYLQKPG QGLEWMGMINPSWGSTSYAQK QSPQLLIYLGSNRASGVPDRFSGSFQGRVTMTRDTSTSTVYMELSS GSGTDFTLKISRVEAEDVGVYYC LRSEDTAVYYCAREAADGFVGEMQDVALPITFGGGTKVEIK [SEQ RYFDLWGRGTLVTVSS [SEQ ID ID NO: 8] NO: 7]CDR1: RSSQSLLYSNGYNYLD CDR1: YTFSDYYMH [SEQ ID [SEQ ID NO: 42]NO: 39](non-Kabat) or DYYMH CDR2: LGSNRAS [SEQ ID NO: 43][SEQ ID NO: 437] CDR3: MQDVALPIT [SEQ ID CDR2: MINPSWGSTSYAQKFQG NO: 44][SEQ ID NO: 40] CDR3: AREAADGFVGERYFDL [SEQ ID NO: 41](non-Kabat) orEAADGFVGERYFDL [SEQ ID NO: 438] scFv ofDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQS Ab4PQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQ DVALPITFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQ

LEWMGMIN PSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWGRGTLVTVSS [SEQ ID NO: 211]QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQ

L EWMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWGRGTLVTVSS GGGGSGGGGSGG GGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCMQDVALPITFG

GTKVEIK [SEQ ID NO: 212] ExemplaryGACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCC nucleotideGGCGAGCCCGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGCT sequenceGTACAGCAACGGCTACAACTACCTGGACTGGTACCTGCAGAAGCC of Ab4CGGCCAGAGCCCCCAGCTGCTGATCTACCTGGGCAGCAACAGGG scFvCCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGACGTGGCCCTGCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCAGCGACTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATGATCAACCCCAGCTGGGGCAGCACCAGCTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCGCCGACGGCTTCGTGGGCGAGAGGTACTTCGACCTGTGGGGCAGGGGCAGCCTGGTGACCGTGAGCAGC [SEQ ID NO: 252]CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCAGCGACTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATGATCAACCCCAGCTGGGGCAGCACCAGCTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCGCCGACGGCTTCGTGGGCGAGAGGTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCCGGCGAGCCCGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGCTGTACAGCAACGGCTACAACTACCTGGACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACCTGGGCAGCAACAGGGCCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGACGTGGCCCTGCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 253] ADI-11807 EVQLVESGGGLVQPGGSLRLSCDIQMTQSPSTLSASVGDRVTITCR [Ab5] AASGFTFGSYWMSWVRQAPGKASQSISSWLAWYQQKPGKAPKL (I07) GLEWVATIKQDGSEKSYVDSVKLIYEASSLESGVPSRFSGSGSGTE GRFTISRDNAKNSLYLQMNSLR FTLTISSLQPDDFATYYCQQSQSYAEDTAVYYCARPLNAGELDVW PPITFGGGTKVEIK [SEQ ID GQGTMVTVSS [SEQ ID NO: 9]NO: 10] CDR1: FTFGSYWMS [SEQ ID CDR1: RASQSISSWLA [SEQ IDNO: 45](non-Kabat) or SYWMS NO: 48] [SEQ ID NO: 181]CDR2: EASSLES [SEQ ID NO: 49] CDR2: TIKQDGSEKSYVDSVKGCDR3: QQSQSYPPIT [SEQ ID [SEQ ID NO: 46] NO: 50]CDR3: ARPLNAGELDV [SEQ ID NO: 47](non-Kabat) orRPLNAGELDV [SEQ ID NO: 182] scFv of I07 scFv Ab5DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPI TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGK

LEWVATIKQDGSEKS YVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS [SEQ ID NO: 188]EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGK

LE WVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS GGGGSGGGGSGGGGSGG GGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQS YPPITFG

GTKVEIK [SEQ ID NO: 213] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCA sequenceGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab5AAGCTGCTGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCC scFvCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGAGCCAGAGCTACCCCCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCACCATCAAGCAGGACGGCAGCGAGAAGAGCTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCCCCTGAACGCCGGCGAGCTGGACGTGTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC [SEQ ID NO: 254]GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCACCATCAAGCAGGACGGCAGCGAGAAGAGCTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCCCCTGAACGCCGGCGAGCTGGACGTGTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGAGCCAGAGCTACCCCCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 255] ADI-11809EVQLVESGGGLVQPGGSLRLSC DIQMTQSPSTLSASVGDRVTITCR [Ab6]AASGFTFPSYWMSWVRQAPGK ASQSISSWLAWYQQKPGKAPKL GLEWVATIKRDGSEKGYVDSVLIYEASSLESGVPSRFSGSGSGTE KGRFTISRDNAKNSLYLQMNSL FTLTISSLQPDDFATYYCQQSQSYRAEDTAVYYCARPLNAGELDV PPITFGGGTKVEIK [SEQ ID WGQGTMVTVSS [SEQ ID NO: 12]NO: 11] CDR1: RASQSISSWLA [SEQ ID CDR1: FTFPSYWMS [SEQ ID NO: 54]NO: 51](non-Kabat) or SYWMS CDR2: EASSLES [SEQ ID NO: 55][SEQ ID NO: 181] CDR3: QQSQSYPPIT [SEQ ID CDR2: TIKRDGSEKGYVDSVKGNO: 56] [SEQ ID NO:52] CDR3: ARPLNAGELDV [SEQ ID NO: 53](non-Kabat) orPLNAGELDV [SEQ ID NO: 439] scFv ofDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLI Ab6YEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPI TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGK

LEWVATIKRDGSEKG YVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS [SEQ ID NO: 214]EVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGK

LE WVATIKRDGSEKGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS GGGGSGGGGSGGGGSGG GGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQS YPPITFG

GTKVEIK [SEQ ID NO: 215] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCA sequenceGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab6AAGCTGCTGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCC scFvCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGAGCCAGAGCTACCCCCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCCCCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCACCATCAAGAGGGACGGCAGCGAGAAGGGCTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCCCCTGAACGCCGGCGAGCTGGACGTGTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC [SEQ ID NO: 256]GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCCCCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCACCATCAAGAGGGACGGCAGCGAGAAGGGCTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCCCCTGAACGCCGGCGAGCTGGACGTGTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGAGGCCAGCAGCCTGGAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGAGCCAGAGCTACCCCCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 257] ADI-11815QVQLVQSGAEVKKPGASVKVS DIQMTQSPSSVSASVGDRVTITC [Ab7]CKASGYTFGTYYMHWVRQAPG RASQGIDSWLAWYQQKPGKAPK QGLEWMGIINPSRGSTVYAQKFLLIYAASSLQSGVPSRFSGSGSGT QGRVTMTRDTSTSTVYMELSSL DFTLTISSLQPEDFATYYCQQAHSRSEDTAVYYCARGAGYDDEDM YPLTFGGGTKVEIK [SEQ ID DVWGKGTTVTVSS [SEQ IDNO: 14] NO: 13] CDR1: RASQGIDSWLA [SEQ ID CDR1: YTFGTYYMH [SEQ IDNO: 60] NO: 57](non-Kabat) or TYYMH CDR2: AASSLQS [SEQ ID NO: 61][SEQ ID NO: 440] CDR3: QQAHSYPLT [SEQ ID CDR2: IINPSRGSTVYAQKFQG NO: 62][SEQ ID NO:58] CDR3: ARGAGYDDEDMDV [SEQ ID NO: 59](non-Kabat) orGAGYDDEDMDV [SEQ ID NO: 441] scFv ofDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLL Ab7IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPL TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQ

LEWMGIINPSRGSTV YAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWGKGTTVTVSS [SEQ ID NO: 216]QVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQ

LEWMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWGKGTTVTVSS GGGGSGGGGSGGGG SGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QAHSYPLTFG

GTKVEIK [SEQ ID NO: 217] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCG sequenceACAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab 7AAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCC scFvAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCCACAGCTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCGGCACCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATCATCAACCCCAGCAGGGGCAGCACCGTGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCGCCGGCTACGACGACGAGGACATGGACGTGTGGGGCAAGGGCACCACCGTGACCGTGAGCAGC [SEQ ID NO: 258]CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCGGCACCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATCATCAACCCCAGCAGGGGCAGCACCGTGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCGCCGGCTACGACGACGAGGACATGGACGTGTGGGGCAAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCGACAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCCACAGCTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO:  259] ADI-11819EVQLVESGGGLVKPGGSLRLSC DIQMTQSPSTLSASVGDRVTITCR [Ab8]AASGFTFSSYAMSWVRQAPGK ASNSISSWLAWYQQKPGKAPKL GLEWVSSISSSSEGIYYADSVKGLIYEASSTKSGVPSRFSGSGSGTE RFTISRDNAKNSLYLQMNSLRA FTLTISSLQPDDFATYYCQQYDDEDTAVYYCAREGGPYYDSSGYF LPTFGGGTKVEIK [SEQ ID VYYGMDVWGQGTTVTVSS NO: 16][SEQ ID NO: 15] CDR1: RASNSISSWLA [SEQ ID CDR1: FTFSSYAMS [SEQ IDNO: 66] NO: 63](non-Kabat) or SYAMS CDR2: EASSTKS [SEQ ID NO: 67][SEQ ID NO: 442] CDR3: QQYDDLPT [SEQ ID CDR2: SISSSSEGIYYADSVKG NO: 68][SEQ ID NO: 64] CDR3: AREGGPYYDSSGYFVYYGMDV[SEQ ID NO: 65](non-Kabat) or EGGPYYDSSGYFVYYGMDV [SEQ ID NO: 443]scFv of DIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLI Ab8YEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPT FG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGK

LEWVSSISSSSEGIYYA DSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS [SEQ ID NO: 218]EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGK

LE WVSSISSSSEGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS GGGGSGGGG SGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFA TYYCQQYDDLPTFG

GTKVEIK [SEQ ID NO: 219] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCAACAGCATCA sequenceGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab8AAGCTGCTGATCTACGAGGCCAGCAGCACCAAGAGCGGCGTGCC scFvCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACGACGACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGAGCAGCATCAGCAGCAGCAGCGAGGGCATCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCCCCTACTACGACAGCAGCGGCTACTTCGTGTACTACGGCATGG ACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC [SEQ ID NO: 260] GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGAGCAGCATCAGCAGCAGCAGCGAGGGCATCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCCCCTACTACGACAGCAGCGGCTACTTCGTGTACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCAACAGCATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGAGGCCAGCAGCACCAAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACGACGACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 261] ADI-11830 EVQLVESGGGLVQPGGSLRLSCDIQMTQSPSSLSASVGDRVTITCR [Ab9] AASGFTFSSYWMSWVRQAPGKASQVIYSYLNWYQQKPGKAPKL GLEWVANINTDGSEVYYVDSV LIYAASSLKSGVPSRFSGSGSGTDKGRFTISRDNAKNSLYLQMNSL FTLTISSLQPEDFATYYCQQVYD RAEDTAVYYCARDVGPGIAYQTPLTFGGGTKVEIK [SEQ ID GHFDYWGQGTLVTVSS [SEQ ID NO: 18] NO: 17]CDR1: RASQVIYSYLN [SEQ ID CDR1: FTFSSYWMS [SEQ ID NO: 72]NO: 69](non-Kabat) or SYWMS CDR2: AASSLKS [SEQ ID NO: 73][SEQ ID NO: 181] CDR3: QQVYDTPLT [SEQ ID CDR2: NINTDGSEVYYVDSVKG NO: 74][SEQ ID NO: 70] CDR3: ARDVGPGIAYQGHFDY [SEQ ID NO: 71](non-Kabat) orDVGPGIAYQGHFDY [SEQ ID NO: 444] scFv ofDIQMTQSPSSLSASVGDRVTITCRASQVIYSYLNWYQQKPGKAPKLLI Ab9YAASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPL TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LEWVANINTDGSEVY YVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDYWGQGTLVTVSS [SEQ ID NO: 220]EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LE WVANINTDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDYWGQGTLVTVSS GGGGSGGGGSGGG GSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQVIYSYLNWYQQKPGKAPKLLIYAASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QVYDTPLTFG

GTKVEIK [SEQ ID NO: 221] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGTGATCT sequenceACAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab9AAGCTGCTGATCTACGCCGCCAGCAGCCTGAAGAGCGGCGTGCCC scFvAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACGACACCCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACACCGACGGCAGCGAGGTGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCCCCGGCATCGCCTACCAGGGCCACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 262]GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACACCGACGGCAGCGAGGTGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCCCCGGCATCGCCTACCAGGGCCACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGTGATCTACAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGAAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACGACACCCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 263] ADI-11835QLQLQESGPGLVKPSETLSLTCT EIVLTQSPATLSLSPGERATLSCR [Ab10]VSGGSISSTDYYWGWIRQPPGK ASHSVYSYLAWYQQKPGQAPRL (I35)GLEWIGSIGYSGTYYNPSLKSRV LIYDASNRATGIPARFSGSGSGTD TISVDTSKNQFSLKLSSVTAADTFTLTISSLEPEDFAVYYCQQYDN AVYYCARETAHDVHGMDVWG LPTFGGGTKVEIK [SEQ IDQGTTVTVSS [SEQ ID NO: 19] NO: 20] CDR1: GSISSTDYYWG [SEQ IDCDR1: RASHSVYSYLA [SEQ ID NO: 75](non-Kabat) or STDYYWG NO: 78][SEQ ID NO: 445] CDR2: DASNRAT [SEQ ID NO: 79] CDR2: SIGYSGTYYNPSLKSCDR3: QQYDNLPT [SEQ ID [SEQ ID NO: 76] NO: 80] CDR3: ARETAHDVHGMDV [SEQID NO: 77](non-Kabat) or ETAHDVHGMDV [SEQ ID NO: 446] scFv ofEIVLTQSPATLSLSPGERATLSCRASHSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPT Ab10 FG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QLQLQESGPGLVKPSETLSLTCTVSGGSISSTDYYWGWIRQPPGK

LEWIGSIGYSGTYYNPS LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQGTTVTVSS [SEQ ID NO: 222]QLQLQESGPGLVKPSETLSLTCTVSGGSISSTDYYWGWIRQPPGK

LE WIGSIGYSGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQGTTVTVSS GGGGSGGGGSGGGGSGGGG SEIVLTQSPATLSLSPGERATLSCRASHSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPT FG

GTKVEIK [SEQ ID NO: 223] ExemplaryGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCC nucleotideGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCACAGCGTGTA sequenceCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCA of Ab10GGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCG scFvCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGACAACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGCTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCACCGACTACTACTGGGGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGTGGATCGGCAGCATCGGCTACAGCGGCACCTACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGGAGACCGCCCACGACGTGCACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC [SEQ ID NO: 264]CAGCTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCACCGACTACTACTGGGGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGTGGATCGGCAGCATCGGCTACAGCGGCACCTACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGGAGACCGCCCACGACGTGCACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCACAGCGTGTACAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGACAACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 265] ADI-10154EVQLLESGGGLVQPGGSLRLSC EIVLTQSPGTLSLSPGERATLSCR [Ab11]AASGFTFSSYAMSWVRQAPGK ASQSVSSSFLAWYQQKPGQAPR GLEWVSAISASGGSTYYADSVKLLIYGASSRATGIPDRFSGSGSGT GRFTISRDNSKNTLYLQMNSLR DFTLTISRLEPEDFAVYYCQQASSAEDTAVYYCARPRAYYDSSGFK SPPTFGGGTKVEIK [SEQ ID VNYGMDVWGQGTTVTVSSNO: 267] [SEQ ID NO: 266] CDR1: RASQSVSSSFLA [SEQ IDCDR1: SYAMS [SEQ ID NO: 304] NO: 307] or FTFSSYAMS [SEQ ID NO: 528]CDR2: GASSRAT [SEQ ID NO: 308] (non-Kabat) CDR3: QQASSSPPT [SEQ IDCDR2: AISASGGSTYYADSVKG NO: 309] [SEQ ID NO: 305] CDR3:PRAYYDSSGFKVNYGMDV [SEQ ID NO: 306] or ARPRAYYDSSGFKVNYGMDV[SEQ ID NO: 529](non-Kabat) scFv ofEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLI Ab11YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQASSSPPT FG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK

LEWVSAISASGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPRAYYDSSGFKVNYGMDVWGQGTTVTVSS [SEQ ID NO: 447]EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK

LE WVSAISASGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPRAYYDSSGFKVNYGMDVWGQGTTVTVSS GGGGSGGGG SGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA VYYCQQASSSPPTFG

GTKVEIK [SEQ ID NO: 448] ExemplaryGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCC nucleotideGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAG sequenceCAGCAGCTTCCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCC of Ab11CAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCC scFvCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGCCAGCAGCAGCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGAGCGCCATCAGCGCCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCCCAGGGCCTACTACGACAGCAGCGGCTTCAAGGTGAACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC [SEQ ID NO: 489]GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGAGCGCCATCAGCGCCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGCCCAGGGCCTACTACGACAGCAGCGGCTTCAAGGTGAACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCAGCTTCCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGCCAGCAGCAGCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 490] ADI-10155 EVQLLESGGGLVQPGGSLRLSCEIVLTQSPGTLSLSPGERATLSCR [Ab 12] AASGFTFSSYAMSWVRQAPGKASQSVSSDYLAWYQQKPGQAPR GLEWVSGISGSGGSTYYADSVK LLIYGASSRATGIPDRFSGSGSGTGRFTISRDNSKNTLYLQMNSLR DFTLTISRLEPEDFAVYYCQQHSS AEDTAVYYCAREGHSSSYYDHAPPTFGGGTKVEIK [SEQ ID AFDIWGQGTMVTVSS [SEQ ID NO: 269] NO: 268]CDR1: RASQSVSSDYLA [SEQ ID CDR1: SYAMS [SEQ ID NO: 310] NO: 313]or FTFSSYAMS [SEQ ID NO: 530] CDR2: GASSRAT [SEQ ID NO: 314] (non-Kabat)CDR3: QQHSSAPPT [SEQ ID CDR2: GISGSGGSTYYADSVKG NO: 315][SEQ ID NO: 311] CDR3: EGHSSSYYDHAFDI [SEQ ID NO: 312] orAREGHSSSYYDHAFDI [SEQ ID NO: 531](non-Kabat) scFv ofEIVLTQSPGTLSLSPGERATLSCRASQSVSSDYLAWYQQKPGQAPRLL Ab 12IYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHSSAPP TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK

LEWVSGISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGHSSSYYDHAFDIWGQGTMVTVSS [SEQ ID NO: 449]EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGK

LE WVSGISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGHSSSYYDHAFDIWGQGTMVTVSS GGGGSGGGGSGGG GSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSDYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQHSSAPPTFG

GTKVEIK [SEQ ID NO: 450] ExemplaryGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCC nucleotideGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAG sequenceCAGCGACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCC of Ab12CCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCC scFvCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCACAGCAGCGCCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGAGCGGCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCCACAGCAGCAGCTACTACGACCACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC [SEQ ID NO: 491]GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGAGCGGCATCAGCGGCAGCGGCGGCAGCACCTACTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCCACAGCAGCAGCTACTACGACCACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCGACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCACAGCAGCGCCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 492] ADI-10157QVQLQESGPGLVKPSETLSLTCT EIVMTQSPATLSVSPGERATLSCR [Ab13]VSGGSISSYYWSWIRQPPGKGLE ASQSVSSNLAWYQQKPGQAPRL WIGSIYYSGSTNYNPSLKSRVTISLIYGASTRATGIPARFSGSGSGTE VDTSKNQFSLKLSSVTAADTAV FTLTISSLQSEDFAVYYCQQYTVYYCARVGGVYSTIETYGMDVW YPPTFGGGTKVEIK [SEQ ID GQGTTVTVSS [SEQ ID NO: 270]NO: 271] CDR1: SYYWS [SEQ ID NO: 316] CDR1: RASQSVSSNLA [SEQ IDor GSISSYYWS [SEQ ID NO: 532] NO: 319] (non-Kabat) CDR2: GASTRAT [SEQ IDCDR2: SIYYSGSTNYNPSLKS NO: 320] [SEQ ID NO: 317] CDR3: QQYTVYPPT [SEQ IDCDR3: VGGVYSTIETYGMDV NO: 321] [SEQ ID NO: 318] orARVGGVYSTIETYGMDV [SEQ ID NO: 533](non-Kabat) scFv ofEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLI Ab13YGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTVYPP TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGK

LEWIGSIYYSGSTNYNPSL KSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVGGVYSTIETYGMDVWGQGTTVTVSS [SEQ ID NO: 451]QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGK

LEWI GSIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVGGVYSTIETYGMDVWGQGTTVTVSS GGGGSGGGGSGGGGSGG GGSEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYTV YPPTFG

GTKVEIK [SEQ ID NO: 452] ExemplaryGAGATCGTGATGACCCAGAGCCCCGCCACCCTGAGCGTGAGCCCC nucleotideGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAG sequenceCAGCAACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCA of Ab13GGCTGCTGATCTACGGCGCCAGCACCAGGGCCACCGGCATCCCCG scFvCCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACACCGTGTACCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCTACTACTGGAGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGTGGATCGGCAGCATCTACTACAGCGGCAGCACCAACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGGTGGGCGGCGTGTACAGCACCATCGAGACCTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC [SEQ ID NO: 493]CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCTACTACTGGAGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGTGGATCGGCAGCATCTACTACAGCGGCAGCACCAACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGGTGGGCGGCGTGTACAGCACCATCGAGACCTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGATGACCCAGAGCCCCGCCACCCTGAGCGTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCAACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCACCAGGGCCACCGGCATCCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACACCGTGTACCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO:494] ADI-10158QVQLQQWGAGLLKPSETLSLTC EIVMTQSPATLSLSPGERATLSCR [Ab14]AVYGGSFSGYYWSWIRQPPGK ASQSVSSYLAWYQQKPGQAPRL GLEWIGEIDHSGSTNYNPSLKSRLIYDASNRATGIPARFSGSGSGTD VTISVDTSKNQFSLKLSSVTAADFTLTISSLEPEDFAVYYCQQDHNF TAVYYCARQGIHGLRYFDLWG PYTFGGGTKVEIK [SEQ IDRGTLVTVSS [SEQ ID NO: 272] NO: 273] CDR1: GYYWS [SEQ ID NO: 322]CDR1: RASQSVSSYLA [SEQ ID or GSFSGYYWS [SEQ ID NO: 534] NO: 325](non-Kabat) CDR2: DASNRAT [SEQ ID CDR2: EIDHSGSTNYNPSLKS NO: 326][SEQ ID NO: 323] CDR3: QQDHNFPYT [SEQ ID CDR3: QGIHGLRYFDL [SEQ IDNO: 327] NO: 324] or ARQGIHGLRYFDL [SEQ ID NO: 535](non-Kabat) scFv ofEIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLI Ab14YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQDHNFPY TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGK

LEWIGEIDHSGSTNYN PSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARQGIHGLRYFDLWGRGTLVTVSS [SEQ ID NO: 453]QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGK

LE WIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARQGIHGLRYFDLWGRGTLVTVSS GGGGSGGGGSGGGGSGGGG SEIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQDHNFP YTFG

GTKVEIK [SEQ ID NO: 454] ExemplaryGAGATCGTGATGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCC nucleotideGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAG sequenceCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGGCCCCA of Ab14GGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCG scFvCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGACCACAACTTCCCCTACACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGTGGATCGGCGAGATCGACCACAGCGGCAGCACCAACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGCAGGGCATCCACGGCCTGAGGTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 495]CAGGTGCAGCTGCAGCAGTGGGGCGCCGGCCTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGCAGCTTCAGCGGCTACTACTGGAGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGTGGATCGGCGAGATCGACCACAGCGGCAGCACCAACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGCAGGGCATCCACGGCCTGAGGTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGATGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGACCACAACTTCCCCTACACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 496] ADI-10160EVQLVESGGGLVQPGGSLRLSC DIQMTQSPSSLSASVGDRVTITCR [Ab15]AASGFTFSSYWMSWVRQAPGK ASQSISSYLNWYQQKPGKAPKLL GLEWVANINQDGSEKYYVDSVIYAASSLQSGVPSRFSGSGSGTDF KGRFTISRDNAKNSLYLQMNSL TLTISSLQPEDFATYYCQQQYVTRAEDTAVYYCAREANYYGNVG PITFGGGTKVEIK [SEQ ID DDYWGQGTLVTVSS [SEQ IDNO: 275] NO: 274] CDR1: RASQSISSYLN [SEQ ID CDR1: SYWMS [SEQ ID NO: 328]NO: 331] or FTFSSYWMS [SEQ ID NO: 536] CDR2: AASSLQS [SEQ ID NO: 332](non-Kabat) CDR3: QQQYVTPIT [SEQ ID CDR2: NINQDGSEKYYVDSVKG NO: 333][SEQ ID NO: 329] CDR3: EANYYGNVGDDY [SEQ ID NO: 330] orAREANYYGNVGDDY [SEQ ID NO: 537](non-Kabat) scFv ofDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI Ab15YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQYVTPIT FG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LEWVANINQDGSEKY YVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREANYYGNVGDDYWGQGTLVTVSS [SEQ ID NO: 455]EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LE WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREANYYGNVGDDYWGQGTLVTVSS GGGGSGGGGSGGGG SGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ QYVTPITFG

GTKVEIK [SEQ ID NO: 456] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCA sequenceGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab15AAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCC scFvAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCAGTACGTGACCCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCAACTACTACGGCAACGTGGGCGACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 497]GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCAACTACTACGGCAACGTGGGCGACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCAGTACGTGACCCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 498] ADI-10161EVQLVESGGGLVQPGGSLRLSC DIQMTQSPSSVSASVGDRVTITC [Ab16]AASGFTFSSYWMSWVRQAPGK RASQGISSWLAWYQQKPGKAPK GLEWVANINQDGSEKYYVDSVLLIYAASNLQSGVPSRFSGSGSGT KGRFTISRDNAKNSLYLQMNSL DFTLTISSLQPEDFATYYCQQKLSRAEDTAVYYCAREGGDSWYHA LPLTFGGGTKVEIK [SEQ ID FDIWGQGTMVTVSS [SEQ IDNO: 277] NO: 276] CDR1: RASQGISSWLA [SEQ ID CDR1: SYWMS [SEQ ID NO: 334]NO: 337] or FTFSSYWMS [SEQ ID NO: 538] CDR2: AASNLQS [SEQ ID (non-Kabat)NO: 338] CDR2: NINQDGSEKYYVDSVKG CDR3: QQKLSLPLT [SEQ ID[SEQ ID NO: 335] NO: 339] CDR3: EGGDSWYHAFDI [SEQ ID NO: 336] orAREGGDSWYHAFDI [SEQ ID NO: 539](non-Kabat) scFv ofDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLL Ab16IYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKLSLPL TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LEWVANINQDGSEKY YVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGDSWYHAFDIWGQGTMVTVSS [SEQ ID NO: 457]EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LE WVANINQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGDSWYHAFDIWGQGTMVTVSS GGGGSGGGGSGGGG SGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQKLSLPLTFG

GTKVEIK [SEQ ID NO: 458] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCA sequenceGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab16AAGCTGCTGATCTACGCCGCCAGCAACCTGCAGAGCGGCGTGCCC scFvAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAAGCTGAGCCTGCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCGACAGCTGGTACCACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC [SEQ ID NO: 499]GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGGCGGCGACAGCTGGTACCACGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAACCTGCAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAAGCTGAGCCTGCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 500] ADI-10163QVQLQESGPGLVKPSQTLSLTCT DIQMTQSPSSLSASVGDRVTITCR [Ab17]VSGGSISSGGYYWSWIRQHPGK ASQSISSYLNWYQQKPGKAPKLL GLEWIGSIYYSGSTYYNPSLKSRIYGASSLQSGVPSRFSGSGSGTDF VTISVDTSKNQFSLKLSSVTAADTLTISSLQPEDFATYYCQQVYSAP TAVYYCARDRLDYSYNYGMDVFTFGGGTKVEIK [SEQ ID NO: 279] WGQGTTVTVSS [SEQ IDCDR1: RASQSISSYLN [SEQ ID NO: 278] NO: 343] CDR1: SGGYYWS [SEQ IDCDR2: GASSLQS [SEQ ID NO: 344] NO: 340] or GSISSGGYYWS [SEQCDR3: QQVYSAPFT [SEQ ID ID NO: 540](non-Kabat) NO: 345]CDR2: SIYYSGSTYYNPSLKS [SEQ ID NO: 341] CDR3: DRLDYSYNYGMDV [SEQID NO: 342] or ARDRLDYSYNYGMDV [SEQ ID NO: 541](non-Kabat) scFv ofDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI Ab17YGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSAPF TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGK

LEWIGSIYYSGSTYYN PSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRLDYSYNYGMDVWGQGTTVTVSS [SEQ ID NO: 459]QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGK

L EWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRLDYSYNYGMDVWGQGTTVTVSS GGGGSGGGGSGGGGS GGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQV YSAPFTFG

GTKVEIK [SEQ ID NO: 460] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCA sequenceGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab17AAGCTGCTGATCTACGGCGCCAGCAGCCTGCAGAGCGGCGTGCCC scFvAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACAGCGCCCCCTTCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCGGCGGCTACTACTGGAGCTGGATCAGGCAGCACCCCGGCAAGTGCCTGGAGTGGATCGGCAGCATCTACTACAGCGGCAGCACCTACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGGACAGGCTGGACTACAGCTACAACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC [SEQ ID NO: 501]CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCGGCGGCTACTACTGGAGCTGGATCAGGCAGCACCCCGGCAAGTGCCTGGAGTGGATCGGCAGCATCTACTACAGCGGCAGCACCTACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGGACAGGCTGGACTACAGCTACAACTACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGGCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACAGCGCCCCCTTCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 502] ADI-10164QVQLQESGPGLVKPSETLSLTCA EIVLTQSPATLSLSPGERATLSCR [Ab18]VSGYSISSGYYWGWIRQPPGKG ASQSVSSYLAWYQQKPGQAPRL LEWIGSIYHSGSTNYNPSLKSRVLIYDASNRATGIPARFSGSGSGTD TISVDTSKNQFSLKLSSVTAADT FTLTISSLEPEDFAVYYCQQVDNAVYYCARLPPWFGFSYFDLWG YPPTFGGGTKVEIK [SEQ ID RGTLVTVSS [SEQ ID NO: 280]NO: 281] CDR1: SGYYWG [SEQ ID CDR1: RASQSVSSYLA [SEQ IDNO: 346] or YSISSGYYWG [SEQ NO: 349] ID NO: 542](non-Kabat)CDR2: DASNRAT [SEQ ID CDR2: SIYHSGSTNYNPSLKS NO: 350] [SEQ ID NO: 347]CDR3: QQVDNYPPT [SEQ ID CDR3: LPPWFGFSYFDL [SEQ ID NO: 351]NO: 348] or ARLPPWFGFSYFDL [SEQ ID NO: 543](non-Kabat) scFv ofEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLI Ab18YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQVDNYPP TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWGWIRQPPGK

LEWIGSIYHSGSTNYNP SLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPPWFGFSYFDLWGRGTLVTVSS [SEQ ID NO: 461]QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWGWIRQPPGK

LE WIGSIYHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLPPWFGFSYFDLWGRGTLVTVSS GGGGSGGGGSGGGGSGGG GSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQVDNY PPTFG

GTKVEIK [SEQ ID NO: 462] ExemplaryGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCC nucleotideGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAG sequenceCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCA of Ab 18GGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCG scFvCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGTGGACAACTACCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGAGCGGCTACAGCATCAGCAGCGGCTACTACTGGGGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGTGGATCGGCAGCATCTACCACAGCGGCAGCACCAACTACAACCCCAGCCTGAAGAGCAGGG1GACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGCTGCCCCCCTGGTTCGGCTTCAGCTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 503]CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGAGCGGCTACAGCATCAGCAGCGGCTACTACTGGGGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGTGGATCGGCAGCATCTACCACAGCGGCAGCACCAACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGCTGCCCCCCTGGTTCGGCTTCAGCTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGTGGACAACTACCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 504] ADI-10165EVQLVESGGGLVQPGGSLRLSC DIQMTQSPSSLSASVGDRVTITCR [Ab19]AASGFTFSSYWMSWVRQAPGK ASQSISSYLNWYQQKPGKAPKLL GLEWVANIKQDGSEKYYVDSVIYAASSLQSGVPSRFSGSGSGTDF KGRFTISRDNAKNSLYLQMNSL TLTISSLQPEDFATYYCQQVYDTRAEDTAVYYCARDVGPGIAYQ PLTFGGGTKVEIK [SEQ ID GHFDYWGQGTLVTVSS [SEQ IDNO: 283] NO: 282] CDR1: RASQSISSYLN [SEQ ID CDR1: SYWMS [SEQ ID NO: 352]NO: 355] or FTFSSYWMS [SEQ ID NO: 544] CDR2: AASSLQS [SEQ ID NO: 356](non-Kabat) CDR3: QQVYDTPLT [SEQ ID CDR2: NIKQDGSEKYYVDSVKG NO: 357][SEQ ID NO: 353] CDR3: DVGPGIAYQGHFDY [SEQ ID NO: 354] orARDVGPGIAYQGHFDY [SEQ ID NO: 545](non-Kabat) scFv ofDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI Ab19YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPL TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LEWVANIKQDGSEKY YVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDYWGQGTLVTVSS [SEQ ID NO: 463]EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LE WVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDYWGQGTLVTVSS GGGGSGGGGSGGG GSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QVYDTPLTFG

GTKVEIK [SEQ ID NO: 464] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCA sequenceGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab19AAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCC scFvAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACGACACCCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCCCCGGCATCGCCTACCAGGGCCACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 505]GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAAGCAGGACGGCAGCGAGAAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCCCCGGCATCGCCTACCAGGGCCACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACGACACCCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG[SEQ ID NO: 506] ADI-10167QLQLQESGPGLVKPSETLSLTCT EIVLTQSPATLSLSPGERATLSCR [Ab20]VSGGSISSSSYYWGWIRQPPGK ASQSVSSYLAWYQQKPGQAPRL GLEWIGSIYYSGSTYYNPSLKSRLIYDASNRATGIPARFSGSGSGTD VTISVDTSKNQFSLKLSSVTAAD FTLTISSLEPEDFAVYYCQQYDNTAVYYCARETAHDVHGMDVW LPTFGGGTKVEIK [SEQ ID GQGTTVTVSS [SEQ ID NO: 284]NO: 285] CDR1: SSSYYWG [SEQ ID CDR1: RASQSVSSYLA [SEQ IDNO: 358] or GSISSSSYYWG [SEQ NO: 361] ID NO: 546](non-Kabat)CDR2: DASNRAT [SEQ ID CDR2: SIYYSGSTYYNPSLKS NO: 362] [SEQ ID NO: 359]CDR3: QQYDNLPT [SEQ ID CDR3: ETAHDVHGMDV [SEQ ID NO: 363]NO: 360] or ARETAHDVHGMDV [SEQ ID NO: 547](non-Kabat) scFv ofEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLI Ab20YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPT FG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGK

LEWIGSIYYSGSTYYNP SLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQGTTVTVSS [SEQ ID NO: 465]QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGK

LE WIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQGTTVTVSS GGGGSGGGGSGGGGSGGG GSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLP TFG

GTKVEIK [SEQ ID NO: 466] ExemplaryGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCC nucleotideGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAG sequenceCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCA of Ab20GGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCG scFvCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGACAACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGCTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCAGCAGCTACTACTGGGGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGTGGATCGGCAGCATCTACTACAGCGGCAGCACCTACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGGAGACCGCCCACGACGTGCACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC [SEQ ID NO: 507]CAGCTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCAGCAGCTACTACTGGGGCTGGATCAGGCAGCCCCCCGGCAAGTGCCTGGAGTGGATCGGCAGCATCTACTACAGCGGCAGCACCTACTACAACCCCAGCCTGAAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAGGGAGACCGCCCACGACGTGCACGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACCGfGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGACGCCAGCAACAGGGCCACCGGCATCCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACGACAACCTGCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 508] ADI-10168QVQLVQSGAEVKKPGSSVKVSC EIVLTQSPATLSLSPGERATLSCR [Ab21]KASGGTFSSYAISWVRQAPGQG ASQSVSSYLAWYQQKPGQAPRL LEWMGSIIPIFGTANYAQKFQGRLIYDASKRATGIPARFSGSGSGTD VTITADESTSTAYMELSSLRSEDFTLTISSLEPEDFAVYYCQQSSNH TAVYYCAREVGYGWYTKIAFDI PSTFGGGTKVEIK [SEQ IDWGQGTMVTVSS [SEQ ID NO: 287] NO: 286] CDR1: RASQSVSSYLA [SEQ IDCDR1: SYAIS [SEQ ID NO: 364] or NO: 367] GTFSSYAIS [SEQ ID NO: 548]CDR2: DASKRAT [SEQ ID (non-Kabat) NO: 368] CDR2: SIIPIFGTANYAQKFQGCDR3: QQSSNHPST [SEQ ID [SEQ ID NO: 365] NO: 369]CDR3: EVGYGWYTKIAFDI [SEQ ID NO: 366] or AREVGYGWYTKIAFDI [SEQ IDNO: 549](non-Kabat) scFv ofEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLI Ab21YDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNHPST FG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQ

LEWMGSIIPIFGTANYA QKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREVGYGWYTKIAFDIWGQGTMVTVSS [SEQ ID NO: 467]QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQ

LE WMGSIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREVGYGWYTKIAFDIWGQGTMVTVSS GGGGSGGGGSGGGGS GGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQS SNHPSTFG

GTKVEIK [SEQ ID NO: 468] ExemplaryGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCC nucleotideGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAG sequenceCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCA of Ab21GGCTGCTGATCTACGACGCCAGCAAGAGGGCCACCGGCATCCCC scFvGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCAGCAACCACCCCAGCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCGGCACCTTCAGCAGCTACGCCATCAGCTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCAGCATCATCCCCATCTTCGGCACCGCCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGTGGGCTACGGCTGGTACACCAAGATCGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC [SEQ ID NO: 509]CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCGGCACCTTCAGCAGCTACGCCATCAGCTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCAGCATCATCCCCATCTTCGGCACCGCCAACTACGCCCAGAAGTTCCAGGGCAGGGTGACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGTGGGCTACGGCTGGTACACCAAGATCGCCTTCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGACGCCAGCAAGAGGGCCACCGGCATCCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCAGCAACCACCCCAGCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 510] ADI-10173QVQLVQSGAEVKKPGASVKVS DIVMTQSPLSLPVTPGEPASISCR [Ab22]CKASGYTFTSYYMHWVRQAPG SSQSLLHSNGYNYLDWYLQKPG QGLEWMGIINPSGGSTTYAQKFQSPQLLIYLGSNRASGVPDRFSGS QGRVTMTRDTSTSTVYMELSSL GSGTDFTLKISRVEAEDVGVYYCRSEDTAVYYCAREAADGFVGE MQALGVPLTFGGGTKVEIK [SEQ RYFDLWGRGTLVTVSS [SEQ IDID NO: 289] NO: 288] CDR1: RSSQSLLHSNGYNYLD CDR1: SYYMH [SEQ ID NO: 370][SEQ ID NO: 373] or YTFTSYYMH [SEQ ID NO: 550]CDR2: LGSNRAS [SEQ ID NO: 374] (non-Kabat) CDR3: MQALGVPLT [SEQ IDCDR2: IINPSGGSTTYAQKFQG NO: 375] [SEQ ID NO: 371] CDR3: EAADGFVGERYFDL[SEQ ID NO: 372] or AREAADGFVGERYFDL [SEQ ID NO: 551](non-Kabat) scFv ofDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQS Ab22PQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQ ALGVPLTFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQ

LEWMGIIN PSGGSTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWGRGTLVTVSS [SEQ ID NO: 469]QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQ

L EWMGIINPSGGSTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWGRGTLVTVSS GGGGSGGGGSGGG GSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDV GVYYCMQALGVPLTFG

GTKVEIK [SEQ ID NO: 470] ExemplaryGACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCC nucleotideGGCGAGCCCGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGCT sequenceGCACAGCAACGGCTACAACTACCTGGACTGGTACCTGCAGAAGC of Ab22CCGGCCAGAGCCCCCAGCTGCTGATCTACCTGGGCAGCAACAGG scFvGCCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCTGGGCGTGCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATCATCAACCCCAGCGGCGGCAGCACCACCTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCGCCGACGGCTTCGTGGGCGAGAGGTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 511]CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATCATCAACCCCAGCGGCGGCAGCACCACCTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCGCCGACGGCTTCGTGGGCGAGAGGTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCCGGCGAGCCCGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGCTGCACAGCAACGGCTACAACTACCTGGACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACCTGGGCAGCAACAGGGCCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCTGGGCGTGCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 512] ADI-11802 QVQLVQSGAEVKKPGASVKVSDIVMTQSPLSLPVTPGEPASISCR [Ab23] CKASGYTFSGYYMHWVRQAPGSSQSLLYSNGYNYLDWYLQKPG QGLEWMGMINPYGGSTRYAQK QSPQLLIYLGSNRASGVPDRFSGSFQGRVTMTRDTSTSTVYMELSS GSGTDFTLKISRVEAEDVGVYYC LRSEDTAVYYCAREAADGFVGEMQDVALPITFGGGTKVEIK [SEQ RYFDLWGRGTLVTVSS [SEQ ID ID NO:291] NO: 290]CDR1: RSSQSLLYSNGYNYLD CDR1: GYYMH [SEQ ID NO: 376] [SEQ ID NO: 379]or YTFSGYYMH [SEQ ID CDR2: LGSNRAS [SEQ ID NO: 380] NO: 552](non-Kabat)CDR3: MQDVALPIT [SEQ ID CDR2: MINPYGGSTRYAQKFQG NO: 381][SEQ ID NO: 377] CDR3: EAADGFVGERYFDL [SEQ ID NO: 378] orAREAADGFVGERYFDL [SEQ ID NO: 553](non-Kabat) scFv ofDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQS Ab23PQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQ DVALPITFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKVSCKASGYTFSGYYMHWVRQAPGQ

LEWMGMIN PYGGSTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWGRGTLVTVSS [SEQ ID NO: 471]QVQLVQSGAEVKKPGASVKVSCKASGYTFSGYYMHWVRQAPGQ

L EWMGMINPYGGSTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWGRGTLVTVSS GGGGSGGGGSGG GGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAED VGVYYCMQDVALPITFG

GTKVEIK [SEQ ID NO: 472] ExemplaryGACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCC nucleotideGGCGAGCCCGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGCT sequenceGTACAGCAACGGCTACAACTACCTGGACTGGTACCTGCAGAAGCC of Ab23CGGCCAGAGCCCCCAGCTGCTGATCTACCTGGGCAGCAACAGGG scFvCCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGACGTGGCCCTGCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCAGCGGCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATGATCAACCCCTACGGCGGCAGCACCAGGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCGCCGACGGCTTCGTGGGCGAGAGGTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 513]CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCAGCGGCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATGATCAACCCCTACGGCGGCAGCACCAGGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCGCCGACGGCTTCGTGGGCGAGAGGTACTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCCCCTGAGCCTGCCCGTGACCCCCGGCGAGCCCGCCAGCATCAGCTGCAGGAGCAGCCAGAGCCTGCTGTACAGCAACGGCTACAACTACCTGGACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACCTGGGCAGCAACAGGGCCAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGACGTGGCCCTGCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 514] ADI-11812 QVQLVQSGAEVKKPGASVKVSDIQMTQSPSSVSASVGDRVTITC [Ab24] CKASGYTFEIYYMHWVRQAPGRASQGIDSWLAWYQQKPGKAPK QGLEWMGIINPSSGSTVYAQKF LLIYAASSLQSGVPSRFSGSGSGTQGRVTMTRDTSTSTVYMELSSL DFTLTISSLQPEDFATYYCQQAHS RSEDTAVYYCARGAGYDDEDMYPLTFGGGTKVEIK [SEQ ID DVWGKGTTVTVSS [SEQ ID NO: 293] NO: 292]CDR1: RASQGIDSWLA [SEQ ID CDR1: IYYMH [SEQ ID NO: 382] NO: 385]or YTFEIYYMH [SEQ ID NO: 554] CDR2: AASSLQS [SEQ ID NO: 386] (non-Kabat)CDR3: QQAHSYPLT [SEQ ID CDR2: IINPSSGSTVYAQKFQG NO: 387][SEQ ID NO: 383] CDR3: GAGYDDEDMDV [SEQ ID NO: 384] orARGAGYDDEDMDV [SEQ ID NO: 555](non-Kabat) scFv ofDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLL Ab24IYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPL TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKVSCKASGYTFEIYYMHWVRQAPGQ

LEWMGIINPSSGSTV YAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWGKGTTVTVSS [SEQ ID NO: 473]QVQLVQSGAEVKKPGASVKVSCKASGYTFEIYYMHWVRQAPGQ

L EWMGIINPSSGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWGKGTTVTVSS GGGGSGGGGSGGGGS GGGGSDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ AHSYPLTFG

GTKVEIK [SEQ ID NO: 474] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCG sequenceACAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab24AAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCC scFvAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCCACAGCTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCGAGATCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATCATCAACCCCAGCAGCGGCAGCACCGTGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCGCCGGCTACGACGACGAGGACATGGACGTGTGGGGCAAGGGCACCACCGTGACCGTGAGCAGC [SEQ ID NO: 515]CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCGAGATCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCATCATCAACCCCAGCAGCGGCAGCACCGTGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCGCCGGCTACGACGACGAGGACATGGACGTGTGGGGCAAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCGACAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCCACAGCTACCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 516] ADI-11825EVQLVESGGGLVQPGGSLRLSC DIQMTQSPSSLSASVGDRVTITCR [Ab25]AASGFTFGGYWMSWVRQAPGK ASQSIYNYLNWYQQKPGKAPKL GLEWVANINQDGSEEYYVDSVLIYAASNLHSGVPSRFSGSGSGT KGRFTISRDNAKNSLYLQMNSL DFTLTISSLQPEDFATYYCQQAFHRAEDTAVYYCAREANYYGNVG VPITFGGGTKVEIK [SEQ ID DDYWGQGTLVTVSS [SEQ IDNO: 295] NO: 294] CDR1: RASQSIYNYLN [SEQ ID CDR1: GYWMS [SEQ ID NO: 388]NO: 391] or FTFGGYWMS [SEQ ID CDR2: AASNLHS [SEQ ID NO: 556](non-Kabat)NO: 392] CDR2: NINQDGSEEYYVDSVKG CDR3: QQAFHVPIT [SEQ ID[SEQ ID NO: 389] NO: 393] CDR3: EANYYGNVGDDY [SEQ ID NO: 390] orAREANYYGNVGDDY [SEQ ID NO: 557](non-Kabat) scFv ofDIQMTQSPSSLSASVGDRVTITCRASQSIYNYLNWYQQKPGKAPKLLI Ab25YAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAFHVPI TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFGGYWMSWVRQAPGK

LEWVANINQDGSEE YYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREANYYGNVGDDYWGQGTLVTVSS [SEQ ID NO: 475]EVQLVESGGGLVQPGGSLRLSCAASGFTFGGYWMSWVRQAPGK

L EWVANINQDGSEEYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREANYYGNVGDDYWGQGTLVTVSS GGGGSGGGGSGGG GSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIYNYLNWYQQKPGKAPKLLIYAASNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAFHVPITFG

GTKVEIK [SEQ ID NO: 476] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCT sequenceACAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab25AAGCTGCTGATCTACGCCGCCAGCAACCTGCACAGCGGCGTGCCC scFvAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTTCCACGTGCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGGCGGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGGAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCAACTACTACGGCAACGTGGGCGACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 517]GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCGGCGGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGGAGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCAACTACTACGGCAACGTGGGCGACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCTACAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAACCTGCACAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTTCCACGTGCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 518] ADI-11826EVQLVESGGGLVQPGGSLRLSC DIQMTQSPSSLSASVGDRVTITCR [Ab26]AASGFTFPGYWMSWVRQAPGK ASQSIYNYLNWYQQKPGKAPKL GLEWVANINQDGSEVYYVDSVLIYAASSTQSGVPSRFSGSGSGTD KGRFTISRDNAKNSLYLQMNSL FTLTISSLQPEDFATYYCQQAFHVRAEDTAVYYCAREANYYGNVG PITFGGGTKVEIK [SEQ ID DDYWGQGTLVTVSS [SEQ IDNO: 297] NO: 296] CDR1: RASQSIYNYLN [SEQ ID CDR1: GYWMS [SEQ ID NO: 394]NO: 397] or FTFPGYWMS [SEQ ID NO: 558] CDR2: AASSTQS [SEQ ID NO: 398](non-Kabat) CDR3: QQAFHVPIT [SEQ ID CDR2: NINQDGSEVYYVDSVKG NO: 399][SEQ ID NO: 395] CDR3: EANYYGNVGDDY [SEQ ID NO: 396] orAREANYYGNVGDDY [SEQ ID NO: 559](non-Kabat) scFv ofDIQMTQSPSSLSASVGDRVTITCRASQSIYNYLNWYQQKPGKAPKLLI Ab26YAASSTQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAFHVPIT FG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFPGYWMSWVRQAPGK

LEWVANINQDGSEVY YVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREANYYGNVGDDYWGQGTLVTVSS [SEQ ID NO: 477]EVQLVESGGGLVQPGGSLRLSCAASGFTFPGYWMSWVRQAPGK

LE WVANINQDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREANYYGNVGDDYWGQGTLVTVSS GGGGSGGGGSGGGG SGGGGSDIQMTQSPSSLSASVGDRTITCRASQSIYNYLNWYQQKPGKAPKLLIYAASSTQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ AFHVPITFG

GTKVEIK [SEQ ID NO: 478] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCT sequenceACAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab26AAGCTGCTGATCTACGCCGCCAGCAGCACCCAGAGCGGCGTGCCC scFvAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTTCCACGTGCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCCCCGGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGGTGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCAACTACTACGGCAACGTGGGCGACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 519]GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCCCCGGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGGTGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGGCCAACTACTACGGCAACGTGGGCGACGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCTACAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCACCCAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTTCCACGTGCCCATCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 520] ADI-11828EVQLVESGGGLVQPGGSLRLSC DIQMTQSPSSLSASVGDRVTITCR [Ab27]AASGFTFSSYWMSWVRQAPGK ASQSIYYYLNWYQQKPGKAPKL GLEWVANINQDGSEVYYVDSVLIYAASSRQSGVPSRFSGSGSGTD KGRFTISRDNAKNSLYLQMNSL FTLTISSLQPEDFATYYCQQVYDRAEDTAVYYCARDVGPGIAYQ TPLTFGGGTKVEIK [SEQ ID GHFDYWGQGTLVTVSS [SEQ IDNO: 299] NO: 298] CDR1: RASQSIYYYLN [SEQ ID CDR1: SYWMS [SEQ ID NO: 400]NO: 403] or FTFSSYWMS [SEQ ID NO: 560] CDR2: AASSRQS [SEQ ID NO: 404](non-Kabat) CDR3: QQVYDTPLT [SEQ ID CDR2: NINQDGSEVYYVDSVKG NO: 405][SEQ ID NO: 401] CDR3: DVGPGIAYQGHFDY [SEQ ID NO: 402] orARDVGPGIAYQGHFDY [SEQ ID NO: 561](non-Kabat) scFv ofDIQMTQSPSSLSASVGDRVTITCRASQSIYYYLNWYQQKPGKAPKLLI Ab27YAASSRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPL TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LEWVANINQDGSEVY YVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDYWGQGTLVTVSS [SEQ ID NO: 479]EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LE WVANINQDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDYWGQGTLVTVSS GGGGSGGGGSGGG GSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSIYYYLNWYQQKPGKAPKLLIYAASSRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QVYDTPLTFG

GTKVEIK [SEQ ID NO: 480] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCT sequenceACTACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab27AAGCTGCTGATCTACGCCGCCAGCAGCAGGCAGAGCGGCGTGCC scFvCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACGACACCCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGGTGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCCCCGGCATCGCCTACCAGGGCCACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 521]GAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACTGGATGAGCTGGGTGAGGCAGGCCCCCGGCAAGTGCCTGGAGTGGGTGGCCAACATCAACCAGGACGGCAGCGAGGTGTACTACGTGGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCCCCGGCATCGCCTACCAGGGCCACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGAGCATCTACTACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCAGGCAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACGACACCCCCCTGACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 522] ADI-11839QVQLVQSGAEVKKPGASVKVS DIQMTQSPSSVSASVGDRVTITCE [Ab28]CKASGYTFSNYYMHWVRQAPG ASKGISSWLAWYQQKPGKAPKL QGLEWMGWINPFSGGTRYAQKLIYAASDLQSGVPSRFSGSGSGT FQGRVTMTRDTSTSTVYMELSS DFTLTISSLQPEDFATYYCQQAFLLRSEDTAVYYCARDVGSSAYY FPPTFGGGTKVEIK [SEQ ID YMDVWGKGTTVTVSS [SEQ IDNO: 301] NO: 300] CDR1: EASKGISSWLA [SEQ ID CDR1: NYYMH [SEQ ID NO: 406]NO: 409] or YTFSNYYMH [SEQ ID CDR2: AASDLQS [SEQ ID NO: 562](non-Kabat)NO: 410] CDR2: WINPFSGGTRYAQKFQG CDR3: QQAFLFPPT [SEQ ID[SEQ ID NO: 407] NO: 411] CDR3: DVGSSAYYYMDV [SEQ ID NO: 408] orARDVGSSAYYYMDV [SEQ ID NO: 563](non-Kabat) scFv ofDIQMTQSPSSVSASVGDRVTITCEASKGISSWLAWYQQKPGKAPKLL Ab28IYAASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAFLFPP TFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYMHWVRQAPGQ

LEWMGWINPFSGGT RYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDVGSSAYYYMDVWGKGTTVTVSS [SEQ ID NO: 481]QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYYMHWVRQAPGQ

L EWMGWINPFSGGTRYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDVGSSAYYYMDVWGKGTTVTVSS GGGGSGGGGSGGG GSGGGGSDIQMTQSPSSVSASVGDRVTITCEASKGISSWLAWYQQKPGKAPKLLIYAASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAFLFPPTFG

GTKVEIK [SEQ ID NO: 482] ExemplaryGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGT nucleotideGGGCGACAGGGTGACCATCACCTGCGAGGCCAGCAAGGGCATCA sequenceGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCC of Ab28AAGCTGCTGATCTACGCCGCCAGCGACCTGCAGAGCGGCGTGCCC scFvAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTTCCTGTTCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCAGCAACTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCTGGATCAACCCCTTCAGCGGCGGCACCAGGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCAGCAGCGCCTACTACTACATGGACGTGTGGGGCAAGGGCACCACCGTGACCGTGAGCAGC [SEQ ID NO: 523]CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCAGCAACTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGTGCCTGGAGTGGATGGGCTGGATCAACCCCTTCAGCGGCGGCACCAGGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACCAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGACGTGGGCAGCAGCGCCTACTACTACATGGACGTGTGGGGCAAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCGTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCGAGGCCAGCAAGGGCATCAGCAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCGACCTGCAGAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGCCTTCCTGTTCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 524] ADI-10152EVQLVQSGAEVKKPGESLKISC EIVLTQSPGTLSLSPGERATLSCR [Ab29]KGSGYSFTSYWIGWVRQMPGK ASQSVSSSFLAWYQQKPGQAPR GLEWMGSIYPGDSDTRYSPSFQLLIYGASSRATGIPDRFSGSGSGT GQVTISADKSISTAYLQWSSLKADFTLTISRLEPEDFAVYYCQQLDS SDTAMYYCARELAYGDYKGGV PPPTFGGGTKVEIK [SEQ IDDYWGQGTLVTVSS [SEQ ID NO: 303] NO: 302] CDR1: RASQSVSSSFLA [SEQ IDCDR1: SYWIG [SEQ ID NO: 412] NO: 415] or YSFTSYWIG [SEQ ID NO: 564]CDR2: GASSRAT [SEQ ID NO: 416] (non-Kabat) CDR3: QQLDSPPPT [SEQ IDCDR2: SIYPGDSDTRYSPSFQG NO: 417] [SEQ ID NO: 413]CDR3: ELAYGDYKGGVDY [SEQ ID NO: 414] or ARELAYGDYKGGVDY [SEQ IDNO: 565](non-Kabat) scFv ofEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLI Ab29YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQLDSPPPT FG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGK

LEWMGSIYPGDSDTRY SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARELAYGDYKGGVDYWGQGTLVTVSS [SEQ ID NO: 483]EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGK

LE WMGSIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARELAYGDYKGGVDYWGQGTLVTVSS GGGGSGGGGSGGGG SGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ LDSPPPTFG

GTKVEIK [SEQ ID NO: 484] ExemplaryGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCC nucleotideGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAG sequenceCAGCAGCTTCCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCC of Ab29CAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCC scFvCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCTGGACAGCCCCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAGGGCAGCGGCTACAGCTTCACCAGCTACTGGATCGGCTGGGTGAGGCAGATGCCCGGCAAGTGCCTGGAGTGGATGGGCAGCATCTACCCCGGCGACAGCGACACCAGGTACAGCCCCAGCTTCCAGGGCCAGGTGACCATCAGCGCCGACAAGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCAGCGACACCGCCATGTACTACTGCGCCAGGGAGCTGGCCTACGGCGACTACAAGGGCGGCGTGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC [SEQ ID NO: 525]GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAGGGCAGCGGCTACAGCTTCACCAGCTACTGGATCGGCTGGGTGAGGCAGATGCCCGGCAAGTGCCTGGAGTGGATGGGCAGCATCTACCCCGGCGACAGCGACACCAGGTACAGCCCCAGCTTCCAGGGCCAGGTGACCATCAGCGCCGACAAGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCAGCGACACCGCCATGTACTACTGCGCCAGGGAGCTGGCCTACGGCGACTACAAGGGCGGCGTGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCAGCTTCCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCTGGACAGCCCCCCCCCCACCTTCGGCTGCGGCACCAAGGTGGAGATCAAG [SEQ ID NO: 526] Clone 280-QVQLVQSGAEVKKPGSSVKVSC DIQLTQSPSSLSASVGDRVTITCR 31-01KASGGTFSDYAISWVRQAPGQG ASQGISSVLAWYQQKPGKAPKL (mut) ofLEWMGRIIPILGVADYAQKFQG LIYDASSLESGVPSRFSGSGSGTD WO2012045752RVTITADKSTRTAYMELSSLRSE FTLTISSLQPEDFATYYCQQFDSSI DTAVYYCARNWADAFDIWGQGTFGQGTKLEIK [SEQ ID NO: 419] TMVTVSS [SEQ ID NO: 418]CDR1: RASQGISSVLA [SEQ ID CDR1: DYAIS [SEQ ID NO: 422] NO: 425]CDR2: RIIPILGVADYAQKFQG CDR2: DASSLES [SEQ ID NO: 426] [SEQ ID NO: 423]CDR3: QQFDSSIT [SEQ ID CDR3: NWADAFDI [SEQ ID NO: 427] NO: 424] scFv ofDIQLTQSPSSLSASVGDRVTITCRASQGISSVLAWYQQKPGKAPKLLI clone 280-YDASSLESGVPSRFSGSGSGTDFTILTISSLQPEDFATYYCQQFDSSITF 31-01 G

GTKLEIK GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPG (mut) ofSSVKVSCKASGGTFSDYAISWVRQAPGQ

LEWMGRIIPILGVADYAQ WO2012045752KFQGRVTITADKSTRTAYMELSSLRSEDTAVYYCARNWADAFDIWGQGTMVTVSS [SEQ ID NO: 485] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAISWVRQAPGQ

LE WMGRIIPILGVADYAQKFQGRVTITADKSTRTAYMELSSLRSEDTAVYYCARNWADAFDIWGQGTMVTVSS GGGGSGGGGSGGGGSGGGG SDIQLTQSPSSLSASVGDRVTITCRASQGISSVLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFDSSITF G

GTKLEIK [SEQ ID NO: 486] lintuzumab QVQLVQSGAEVKKPGSSVKVSCDIQMTQSPSSLSASVGDRVTITCR KASGYTFTDYNMHWVRQAPGQ ASESVDNYGISFMNWFQQKPGKGLEWIGYIYPYNGGTGYNQKFK APKLLIYAASNQGSGVPSRFSGS SKATITADESTNTAYMELSSLRSGSGTDFTLTISSLQPDDFATYYCQ EDTAVYYCARGRPAMDYWGQ QSKEVPWTFGQGTKVEIK [SEQGTLVTVSS [SEQ ID NO: 420] ID NO: 421] CDR1: DYNMH [SEQ ID NO: 428]CDR1: RASESVDNYGISFMN CDR2: YIYPYNGGTGYNQKFKS [SEQ ID NO: 431][SEQ ID NO: 429] CDR2: AASNQGS [SEQ ID CDR3: GRPAMDY [SEQ ID NO: 432]NO: 430] CDR3: QQSKEVPWT [SEQ ID NO: 433] scFv ofDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKA lintuzumabPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQS KEVPWTFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQ

LEWIGYIYP YNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS [SEQ ID NO: 487]QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQ

L EWIGYIYPYNGGTGYNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGG SDIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQ SKEVPWTFG

GTKVEIK [SEQ ID NO: 488]An Antigen Binding Site that Binds an Epitope on an Extracellular Domainof Human CD33 and/or Cynomolgus/Rhesus (Cyno) CD33

In one aspect, the invention provides an antigen binding site includinga heavy chain variable domain that binds an epitope on an extracellulardomain of human CD33 and/or Cynomolgus/Rhesus (cyno) CD33.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33 and Cynomolgus/Rhesus (cyno) CD33; the antigen binding siteincludes a heavy chain variable domain including an amino acid sequenceat least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS [SEQ ID NO:1]. In some embodiments, the antibody heavychain variable domain is at least 95% identical to SEQ ID NO:1. In someembodiments, the heavy chain variable domain includes amino acidsequences FTFSSYGMS [SEQ ID NO:21] as the firstcomplementarity-determining region 1 (“CDR1”), NIKQDGSEKYYVDSVKG [SEQ IDNO:22] as the second CDR (“CDR2”), and AREGGPYYDSSGYFVYYGMDV [SEQ IDNO:23] as the third CDR (“CDR3”) of SEQ ID NO:1. In some embodiments,the heavy chain variable domain includes amino acid sequences SYGMS [SEQID NO:434] as the first complementarity-determining region 1 (“CDR1”),NIKQDGSEKYYVDSVKG [SEQ ID NO:22] as the second CDR (“CDR2”), andEGGPYYDSSGYFVYYGMDV [SEQ ID NO:435] as the third CDR (“CDR3”) of SEQ IDNO:1. In certain embodiments, the antibody heavy chain variable domainwhich includes an amino acid sequence at least 90% (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the aminoacid sequence of SEQ ID NO:1 is combined with a light chain variabledomain to form an antigen-binding site capable of binding to CD33. Forexample, an antibody heavy chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:1 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESFPTFGGGTKVEIK [SEQ ID NO:2]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:1 can be pairedwith an antibody light chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:2, which includes amino acidsequences RASQSISSWLA [SEQ ID NO:24] as CDR1, DASSLES [SEQ ID NO:25] asCDR2, and QQYESFPT [SEQ ID NO:26] as CDR3 of SEQ ID NO:2.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33 and Cynomolgus/Rhesus (cyno) CD33; the antigen binding siteincludes a heavy chain variable domain including an amino acid sequenceat least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQ GTMVTVSS [SEQ IDNO:3]. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:3. In some embodiments, the heavychain variable domain incorporates amino acid sequences FTFSSYWMS [SEQID NO:27] as CDR1, NIKQDGSEKYYVDSVKG [SEQ ID NO:28] as CDR2, andARPLNAGELDV [SEQ ID NO:29] as CDR3 of SEQ ID NO:3. In some embodiments,the heavy chain variable domain incorporates amino acid sequences SYWMS[SEQ ID NO:181] as CDR1, NIKQDGSEKYYVDSVKG [SEQ ID NO:28] as CDR2, andPLNAGELDV [SEQ ID NO:436] as CDR3 of SEQ ID NO:3. In certainembodiments, the antibody heavy chain variable domain, which includes anamino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:3, is combined with a light chain variable domain to forman antigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the aminoacid sequence of SEQ ID NO:3 can be paired with an antibody light chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLESYPLTFGGGTKVEIK [SEQ ID NO:4]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:3 can be pairedwith an antibody light chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:4, which includes amino acidsequences RASQSISSWLA [SEQ ID NO:30] as CDR1, EASSLES [SEQ ID NO:31] asCDR2, and QQLESYPLT [SEQ ID NO:32] as CDR3 of SEQ ID NO:4.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33 and Cynomolgus/Rhesus (cyno) CD33; the antigen binding siteincludes a heavy chain variable domain including an amino acid sequenceat least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMSWVRQAPGKGLEWVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS [SEQ ID NO:5]. In some embodiments, the antibody heavychain variable domain is at least 95% identical to SEQ ID NO:5. In someembodiments, the heavy chain variable domain incorporates amino acidsequences FTFSKYTMS [SEQ ID NO:33] as CDR1, AIVGSGESTYFADSVKG [SEQ IDNO:34] as CDR2, and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO:35] as CDR3 of SEQID NO:5. In some embodiments, the heavy chain variable domainincorporates amino acid sequences KYTMS [SEQ ID NO:183] as CDR1,AIVGSGESTYFADSVKG [SEQ ID NO:34] as CDR2, and EGGPYYDSSGYFVYYGMDV [SEQID NO:184] as CDR3 of SEQ ID NO:5. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:5 is combinedwith a light chain variable domain to form an antigen-binding sitecapable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:5 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK [SEQ ID NO:6]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:5 can be pairedwith an antibody light chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:6, which includes amino acidsequences RASQSISSWLA [SEQ ID NO:36] as CDR1, KASSLES [SEQ ID NO:37] orKASSLE [SEQ ID NO:185] as CDR2, and QQYDDLPT [SEQ ID NO:38] as CDR3 ofSEQ ID NO:6.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLEWMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYF DLWGRGTLVTVSS[SEQ ID NO:7]. In some embodiments, the antibody heavy chain variabledomain is at least 95% identical to SEQ ID NO:7. In some embodiments,the heavy chain variable domain incorporates amino acid sequencesYTFSDYYMH [SEQ ID NO:39] as CDR1, MINPSWGSTSYAQKFQG [SEQ ID NO:40] asCDR2, and AREAADGFVGERYFDL [SEQ ID NO:41] as CDR3 of SEQ ID NO:7. Insome embodiments, the heavy chain variable domain incorporates aminoacid sequences DYYMH [SEQ ID NO:437] as CDR1, AIVGSGESTYFADSVKG [SEQ IDNO:34] as CDR2, and EAADGFVGERYFDL [SEQ ID NO:438] as CDR3 of SEQ IDNO:7. In certain embodiments, the antibody heavy chain variable domainwhich includes an amino acid sequence at least 90% (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the aminoacid sequence of SEQ ID NO:7 is combined with a light chain variabledomain to form an antigen-binding site capable of binding to CD33. Forexample, an antibody heavy chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:7 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVALPITFGGGTKVEIK [SEQ ID NO:8]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:7 can be pairedwith an antibody light chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:8, which includes amino acidsequences RSSQSLLYSNGYNYLD [SEQ ID NO:42] as CDR1, LGSNRAS [SEQ IDNO:43] as CDR2, and MQDVALPIT [SEQ ID NO:44] as CDR3 of SEQ ID NO:8.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33 and Cynomolgus/Rhesus (cyno) CD33; the antigen binding siteincludes a heavy chain variable domain including an amino acid sequenceat least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKGLEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQ GTMVTVSS [SEQ IDNO:9]. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:9. In some embodiments, the heavychain variable domain incorporates amino acid sequences FTFGSYWMS [SEQID NO:45] as CDR1, TIKQDGSEKSYVDSVKG [SEQ ID NO:46] as CDR2, andARPLNAGELDV [SEQ ID NO:47] as CDR3 of SEQ ID NO:9. In some embodiments,the heavy chain variable domain incorporates amino acid sequences SYWMS[SEQ ID NO:181] as CDR1, TIKQDGSEKSYVDSVKG [SEQ ID NO:46] as CDR2, andRPLNAGELDV [SEQ ID NO:182] as CDR3 of SEQ ID NO:9. In certainembodiments, the antibody heavy chain variable domain which includes anamino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:9 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the aminoacid sequence of SEQ ID NO:9 can be paired with an antibody light chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGGGTKVEIK [SEQ ID NO:10]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:9 can be pairedwith an antibody light chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:10, which includes amino acidsequences RASQSISSWLA [SEQ ID NO:48] as CDR1, EASSLES [SEQ ID NO:49] asCDR2, and QQSQSYPPIT [SEQ ID NO:50] as CDR3 of SEQ ID NO:10.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33 and Cynomolgus/Rhesus (cyno) CD33; the antigen binding siteincludes a heavy chain variable domain including an amino acid sequenceat least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGKGLEWVATIKRDGSEKGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQG TMVTVSS [SEQ IDNO:11]. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:11. In some embodiments, the heavychain variable domain incorporates amino acid sequences FTFPSYWMS [SEQID NO:51] as CDR1, TIKRDGSEKGYVDSVKG [SEQ ID NO:52] as CDR2, andARPLNAGELDV [SEQ ID NO:53] as CDR3 of SEQ ID NO:11. In some embodiments,the heavy chain variable domain incorporates amino acid sequences SYWMS[SEQ ID NO:181] as CDR1, TIKRDGSEKGYVDSVKG [SEQ ID NO:52] as CDR2, andPLNAGELDV [SEQ ID NO:439] as CDR3 of SEQ ID NO:11. In certainembodiments, the antibody heavy chain variable domain which includes anamino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:11 is combined with a light chain variable domain to forman antigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the aminoacid sequence of SEQ ID NO:11 can be paired with an antibody light chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGGGTKVEIK [SEQ ID NO:12]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:11 can be pairedwith an antibody light chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:12, which includes amino acidsequences RASQSISSWLA [SEQ ID NO:54] as CDR1, EASSLES [SEQ ID NO:55] asCDR2, and QQSQSYPPIT [SEQ ID NO:56] as CDR3 of SEQ ID NO:12.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQGLEWMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDV WGKGTTVTVSS [SEQID NO:13]. In some embodiments, the antibody heavy chain variable domainis at least 95% identical to SEQ ID NO:13. In some embodiments, theheavy chain variable domain incorporates amino acid sequences YTFGTYYMH[SEQ ID NO:57] as CDR1, IINPSRGSTVYAQKFQG [SEQ ID NO:58] as CDR2, andARGAGYDDEDMDV [SEQ ID NO:59] as CDR3 of SEQ ID NO:13. In someembodiments, the heavy chain variable domain incorporates amino acidsequences TYYMH [SEQ ID NO:440] as CDR1, TIKRDGSEKGYVDSVKG [SEQ IDNO:52] as CDR2, and GAGYDDEDMDV [SEQ ID NO:441] as CDR3 of SEQ ID NO:13.In certain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:13 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:13 can be paired with an antibody lightchain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFGGGTKVEIK [SEQ ID NO:14]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:13 can be pairedwith an antibody light chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:14, which includes amino acidsequences RASQGIDSWLA [SEQ ID NO:60] as CDR1, AASSLQS [SEQ ID NO:61] asCDR2, and QQAHSYPLT [SEQ ID NO:62] as CDR3 of SEQ ID NO:14.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33 and Cynomolgus/Rhesus (cyno) CD33; the antigen binding siteincludes a heavy chain variable domain including an amino acid sequenceat least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence ofEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSSSEGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYY GMDVWGQGTTVTVSS[SEQ ID NO:15]. In some embodiments, the antibody heavy chain variabledomain is at least 95% identical to SEQ ID NO:15. In some embodiments,the heavy chain variable domain incorporates amino acid sequencesFTFSSYAMS [SEQ ID NO:63] as CDR1, SISSSSEGIYYADSVKG [SEQ ID NO:64] asCDR2, and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO:65] as CDR3 of SEQ ID NO:15.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SYAMS [SEQ ID NO:442] as CDR1, SISSSSEGIYYADSVKG [SEQ IDNO:64] as CDR2, and EGGPYYDSSGYFVYYGMDV [SEQ ID NO:443] as CDR3 of SEQID NO:15. In certain embodiments, the antibody heavy chain variabledomain which includes an amino acid sequence at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:15 is combined with a light chainvariable domain to form an antigen-binding site capable of binding toCD33. For example, an antibody heavy chain variable domain at least 90%(e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:15 can be paired withan antibody light chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK [SEQ ID NO:16]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:15 can be pairedwith an antibody light chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:16, which includes amino acidsequences RASNSISSWLA [SEQ ID NO:66] as CDR1, EASSTKS [SEQ ID NO:67] asCDR2, and QQYDDLPT [SEQ ID NO:68] as CDR3 of SEQ ID NO:16.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33 and Cynomolgus/Rhesus (cyno) CD33; the antigen binding siteincludes a heavy chain variable domain including an amino acid sequenceat least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINTDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFD YWGQGTLVTVSS[SEQ ID NO:17]. In some embodiments, the antibody heavy chain variabledomain is at least 95% identical to SEQ ID NO:17. In some embodiments,the heavy chain variable domain incorporates amino acid sequencesFTFSSYWMS [SEQ ID NO:69] as CDR1, NINTDGSEVYYVDSVKG [SEQ ID NO:70] asCDR2, and ARDVGPGIAYQGHFDY [SEQ ID NO:71] as CDR3 of SEQ ID NO:17. Insome embodiments, the heavy chain variable domain incorporates aminoacid sequences SYWMS [SEQ ID NO:181] as CDR1, NINTDGSEVYYVDSVKG [SEQ IDNO:70] as CDR2, and DVGPGIAYQGHFDY [SEQ ID NO:444] as CDR3 of SEQ IDNO:17. In certain embodiments, the antibody heavy chain variable domainwhich includes an amino acid sequence at least 90% (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the aminoacid sequence of SEQ ID NO:17 is combined with a light chain variabledomain to form an antigen-binding site capable of binding to CD33. Forexample, an antibody heavy chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:17 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence DIQMTQSPSSLSASVGDRVTITCRASQVIYSYLNWYQQKPGKAPKLLIYAASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPLTFGGGTKVEIK [SEQ ID NO:18]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:17 can be pairedwith an antibody light chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:18, which includes amino acidsequences RASQVIYSYLN [SEQ ID NO:72] as CDR1, AASSLKS [SEQ ID NO:73] asCDR2, and QQVYDTPLT [SEQ ID NO:74] as CDR3 of SEQ ID NO:18.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33 and Cynomolgus/Rhesus (cyno) CD33; the antigen binding siteincludes a heavy chain variable domain including an amino acid sequenceat least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence ofQLQLQESGPGLVKPSETLSLTCTVSGGSISSTDYYWGWIRQPPGKGLEWIGSIGYSGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQG TTVTVSS [SEQ IDNO:19]. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:19. In some embodiments, the heavychain variable domain incorporates amino acid sequences GSISSTDYYWG [SEQID NO:75] as CDR1, SIGYSGTYYNPSLKS [SEQ ID NO:76] as CDR2, andARETAHDVHGMDV [SEQ ID NO:77] as CDR3 of SEQ ID NO:19. In someembodiments, the heavy chain variable domain incorporates amino acidsequences STDYYWG [SEQ ID NO:445] as CDR1, SIGYSGTYYNPSLKS [SEQ IDNO:76] as CDR2, and ETAHDVHGMDV [SEQ ID NO:446] as CDR3 of SEQ ID NO:19.In certain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:19 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:19 can be paired with an antibody lightchain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceEIVLTQSPATLSLSPGERATLSCRASHSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPTFGGGTKVEIK [SEQ ID NO:20]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:19 can be pairedwith an antibody light chain variable domain at least 90% (e.g., atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical tothe amino acid sequence of SEQ ID NO:20, which includes amino acidsequences RASHSVYSYLA [SEQ ID NO:78] as CDR1, DASNRAT [SEQ ID NO:79] asCDR2, and QQYDNLPT [SEQ ID NO:80] as CDR3 of SEQ ID NO:20.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:266. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:266. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:304 as CDR1, SEQ ID NO:305 as CDR2, and SEQ IDNO:306 as CDR3 of SEQ ID NO:266. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:528 as CDR1,SEQ ID NO:305 as CDR2, and SEQ ID NO:529 as CDR3 of SEQ ID NO:266. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:266 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:266 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:267. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:266 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:267, which includes amino acid sequences SEQ ID NO:307 as CDR1, SEQID NO:308 as CDR2, and SEQ ID NO:309 as CDR3 of SEQ ID NO:267.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:268. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:268. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:310 as CDR1, SEQ ID NO:311 as CDR2, and SEQ IDNO:312 as CDR3 of SEQ ID NO:268. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:530 as CDR1,SEQ ID NO:311 as CDR2, and SEQ ID NO:531 as CDR3 of SEQ ID NO:268. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:268 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:268 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:269. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:268 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:269, which includes amino acid sequences SEQ ID NO:313 as CDR1, SEQID NO:314 as CDR2, and SEQ ID NO:315 as CDR3 of SEQ ID NO:269.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:270. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:270. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:316 as CDR1, SEQ ID NO:317 as CDR2, and SEQ IDNO:318 as CDR3 of SEQ ID NO:270. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:532 as CDR1,SEQ ID NO:317 as CDR2, and SEQ ID NO:533 as CDR3 of SEQ ID NO:270. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:270 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:270 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:271. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:270 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:271, which includes amino acid sequences SEQ ID NO:319 as CDR1, SEQID NO:320 as CDR2, and SEQ ID NO:321 as CDR3 of SEQ ID NO:271.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:272. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:272. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:322 as CDR1, SEQ ID NO:323 as CDR2, and SEQ IDNO:324 as CDR3 of SEQ ID NO:272. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:534 as CDR1,SEQ ID NO:323 as CDR2, and SEQ ID NO:535 as CDR3 of SEQ ID NO:272. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:272 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:272 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:273. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:272 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:273, which includes amino acid sequences SEQ ID NO:325 as CDR1, SEQID NO:326 as CDR2, and SEQ ID NO:327 as CDR3 of SEQ ID NO:273.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:274. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:274. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:328 as CDR1, SEQ ID NO:329 as CDR2, and SEQ IDNO:330 as CDR3 of SEQ ID NO:274. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:536 as CDR1,SEQ ID NO:329 as CDR2, and SEQ ID NO:537 as CDR3 of SEQ ID NO:274. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:274 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:274 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:275. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:274 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:275, which includes amino acid sequences SEQ ID NO:331 as CDR1, SEQID NO:332 as CDR2, and SEQ ID NO:333 as CDR3 of SEQ ID NO:275.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:276. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:276. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:334 as CDR1, SEQ ID NO:335 as CDR2, and SEQ IDNO:336 as CDR3 of SEQ ID NO:276. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:538 as CDR1,SEQ ID NO:335 as CDR2, and SEQ ID NO:539 as CDR3 of SEQ ID NO:276. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:276 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:276 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:277. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:276 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:277, which includes amino acid sequences SEQ ID NO:337 as CDR1, SEQID NO:338 as CDR2, and SEQ ID NO:339 as CDR3 of SEQ ID NO:277.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:278. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:278. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:340 as CDR1, SEQ ID NO:341 as CDR2, and SEQ IDNO:342 as CDR3 of SEQ ID NO:278. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:540 as CDR1,SEQ ID NO:341 as CDR2, and SEQ ID NO:541 as CDR3 of SEQ ID NO:278. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:278 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:278 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:279. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:278 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:279, which includes amino acid sequences SEQ ID NO:343 as CDR1, SEQID NO:344 as CDR2, and SEQ ID NO:345 as CDR3 of SEQ ID NO:279.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:280. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:280. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:346 as CDR1, SEQ ID NO:347 as CDR2, and SEQ IDNO:348 as CDR3 of SEQ ID NO:280. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:542 as CDR1,SEQ ID NO:347 as CDR2, and SEQ ID NO:543 as CDR3 of SEQ ID NO:280. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:280 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:280 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:281. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:280 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:281, which includes amino acid sequences SEQ ID NO:349 as CDR1, SEQID NO:350 as CDR2, and SEQ ID NO:351 as CDR3 of SEQ ID NO:281.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:282. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:282. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:352 as CDR1, SEQ ID NO:353 as CDR2, and SEQ IDNO:354 as CDR3 of SEQ ID NO:282. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:544 as CDR1,SEQ ID NO:353 as CDR2, and SEQ ID NO:545 as CDR3 of SEQ ID NO:282. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:282 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:282 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:283. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:282 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:283, which includes amino acid sequences SEQ ID NO:355 as CDR1, SEQID NO:356 as CDR2, and SEQ ID NO:357 as CDR3 of SEQ ID NO:283.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:284. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:284. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:358 as CDR1, SEQ ID NO:359 as CDR2, and SEQ IDNO:360 as CDR3 of SEQ ID NO:284. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:546 as CDR1,SEQ ID NO:359 as CDR2, and SEQ ID NO:360 as CDR3 of SEQ ID NO:547. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:284 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:284 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:285. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:284 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:285, which includes amino acid sequences SEQ ID NO:361 as CDR1, SEQID NO:362 as CDR2, and SEQ ID NO:363 as CDR3 of SEQ ID NO:285.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:286. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:286. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:364 as CDR1, SEQ ID NO:365 as CDR2, and SEQ IDNO:366 as CDR3 of SEQ ID NO:286. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:548 as CDR1,SEQ ID NO:365 as CDR2, and SEQ ID NO:549 as CDR3 of SEQ ID NO:286. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:286 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:286 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:287. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:286 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:287, which includes amino acid sequences SEQ ID NO:367 as CDR1, SEQID NO:368 as CDR2, and SEQ ID NO:369 as CDR3 of SEQ ID NO:287.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:288. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:288. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:370 as CDR1, SEQ ID NO:371 as CDR2, and SEQ IDNO:372 as CDR3 of SEQ ID NO:288. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:550 as CDR1,SEQ ID NO:371 as CDR2, and SEQ ID NO:551 as CDR3 of SEQ ID NO:288. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:288 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:288 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:289. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:288 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:289, which includes amino acid sequences SEQ ID NO:373 as CDR1, SEQID NO:374 as CDR2, and SEQ ID NO:375 as CDR3 of SEQ ID NO:289.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:290. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:290. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:376 as CDR1, SEQ ID NO:377 as CDR2, and SEQ IDNO:378 as CDR3 of SEQ ID NO:290. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:552 as CDR1,SEQ ID NO:377 as CDR2, and SEQ ID NO:553 as CDR3 of SEQ ID NO:290. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:290 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:290 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:291. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:290 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:291, which includes amino acid sequences SEQ ID NO:379 as CDR1, SEQID NO:380 as CDR2, and SEQ ID NO:381 as CDR3 of SEQ ID NO:291.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:292. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:292. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:382 as CDR1, SEQ ID NO:383 as CDR2, and SEQ IDNO:384 as CDR3 of SEQ ID NO:292. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:554 as CDR1,SEQ ID NO:383 as CDR2, and SEQ ID NO:555 as CDR3 of SEQ ID NO:292. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:292 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:292 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:293. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:292 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:293, which includes amino acid sequences SEQ ID NO:385 as CDR1, SEQID NO:386 as CDR2, and SEQ ID NO:387 as CDR3 of SEQ ID NO:293.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:294. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:294. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:388 as CDR1, SEQ ID NO:389 as CDR2, and SEQ IDNO:390 as CDR3 of SEQ ID NO:294. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:556 as CDR1,SEQ ID NO:389 as CDR2, and SEQ ID NO:557 as CDR3 of SEQ ID NO:294. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:294 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:294 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:295. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:294 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:295, which includes amino acid sequences SEQ ID NO:391 as CDR1, SEQID NO:392 as CDR2, and SEQ ID NO:393 as CDR3 of SEQ ID NO:295.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:296. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:296. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:394 as CDR1, SEQ ID NO:395 as CDR2, and SEQ IDNO:396 as CDR3 of SEQ ID NO:296. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:558 as CDR1,SEQ ID NO:395 as CDR2, and SEQ ID NO:559 as CDR3 of SEQ ID NO:296. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:296 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:296 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:297. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:296 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:297, which includes amino acid sequences SEQ ID NO:397 as CDR1, SEQID NO:398 as CDR2, and SEQ ID NO:399 as CDR3 of SEQ ID NO:297.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33 and Cynomolgus/Rhesus (cyno) CD33; the antigen binding siteincludes a heavy chain variable domain including an amino acid sequenceat least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence of SEQ ID NO:298. Insome embodiments, the antibody heavy chain variable domain is at least95% identical to SEQ ID NO:298. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:400 as CDR1,SEQ ID NO:401 as CDR2, and SEQ ID NO:402 as CDR3 of SEQ ID NO:298. Insome embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:560 as CDR1, SEQ ID NO:401 as CDR2, and SEQ IDNO:561 as CDR3 of SEQ ID NO:298. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:298 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:298 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:299. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:298 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:299, which includes amino acidsequences SEQ ID NO:403 as CDR1, SEQ ID NO:404 as CDR2, and SEQ IDNO:405 as CDR3 of SEQ ID NO:299.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:300. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:300. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:406 as CDR1, SEQ ID NO:407 as CDR2, and SEQ IDNO:408 as CDR3 of SEQ ID NO:300. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:562 as CDR1,SEQ ID NO:407 as CDR2, and SEQ ID NO:563 as CDR3 of SEQ ID NO:300. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:300 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:300 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:301. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:300 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:301, which includes amino acid sequences SEQ ID NO:409 as CDR1, SEQID NO:410 as CDR2, and SEQ ID NO:411 as CDR3 of SEQ ID NO:301.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope on an extracellular domain of humanCD33; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:302. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:302. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:412 as CDR1, SEQ ID NO:413 as CDR2, and SEQ IDNO:414 as CDR3 of SEQ ID NO:302. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:564 as CDR1,SEQ ID NO:413 as CDR2, and SEQ ID NO:565 as CDR3 of SEQ ID NO:302. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:302 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:302 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:303. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:302 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:303, which includes amino acid sequences SEQ ID NO:415 as CDR1, SEQID NO:416 as CDR2, and SEQ ID NO:417 as CDR3 of SEQ ID NO:303.

An Antigen Binding Site that Recognizes and Binds a ConformationalEpitope on an Extracellular Domain of the Human CD33 and/or theCynomolgus/Rhesus (Cyno) CD33

In one aspect, the present invention provides an antigen binding siteincluding a heavy chain variable domain that recognizes and binds one ormore conformational epitopes on the extracellular domain of the humanCD33 and/or the Cynomolgus/Rhesus (cyno) CD33.

In certain embodiments, the present invention provides an antigenbinding site that recognizes and binds a conformational epitopepartially located in the V domain of human CD33 extracellular domain;the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQ GTMVTVSS [SEQ IDNO:3]. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:3. In some embodiments, the heavychain variable domain incorporates amino acid sequences FTFSSYWMS [SEQID NO:27] as CDR1, NIKQDGSEKYYVDSVKG [SEQ ID NO:28] as CDR2, andARPLNAGELDV [SEQ ID NO:29] as CDR3 of SEQ ID NO:3. In certainembodiments, the antibody heavy chain variable domain which includes anamino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:3 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:3 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLESYPLTFGGGTKVEIK [SEQ ID NO:4]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:3 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:4, which includes amino acid sequences RASQSISSWLA[SEQ ID NO:30] as CDR1, EASSLES [SEQ ID NO:31] as CDR2, and QQLESYPLT[SEQ ID NO:32] as CDR3 of SEQ ID NO:4.

In certain embodiments, the present invention provides an antigenbinding site that recognizes and binds a conformational epitopepartially located in the V domain of the human CD33 extracellulardomain; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof EVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGKGLEWVATIKRDGSEKGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQG TMVTVSS [SEQ IDNO:11]. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:11. In some embodiments, the heavychain variable domain incorporates amino acid sequences FTFPSYWMS [SEQID NO:51] as CDR1, TIKRDGSEKGYVDSVKG [SEQ ID NO:52] as CDR2, andARPLNAGELDV [SEQ ID NO:53] as CDR3 of SEQ ID NO:11. In certainembodiments, the antibody heavy chain variable domain which includes anamino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:11 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:11 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGGGTKVEIK [SEQ ID NO:12]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:11 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:12, which includes amino acid sequencesRASQSISSWLA [SEQ ID NO:54] as CDR1, EASSLES [SEQ ID NO:55] as CDR2, andQQSQSYPPIT [SEQ ID NO:56] as CDR3 of SEQ ID NO:12.

In certain embodiments, the present invention provides an antigenbinding site that recognizes and binds a conformational epitopepartially located in the V domain of the human CD33 extracellulardomain; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSSSEGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYY GMDVWGQGTTVTVSS[SEQ ID NO:15]. In some embodiments, the antibody heavy chain variabledomain is at least 95% identical to SEQ ID NO:15. In some embodiments,the heavy chain variable domain incorporates amino acid sequencesFTFSSYAMS [SEQ ID NO:63] as CDR1, SISSSSEGIYYADSVKG [SEQ ID NO:64] asCDR2, and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO:65] as CDR3 of SEQ ID NO:15.In certain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:15 is combined with a light chain variable domain to forman antigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:15 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK [SEQ ID NO:16]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:15 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:16, which includes amino acid sequencesRASNSISSWLA [SEQ ID NO:66] as CDR1, EASSTKS [SEQ ID NO:67] as CDR2, andQQYDDLPT [SEQ ID NO:68] as CDR3 of SEQ ID NO:16.

In certain embodiments, the present invention provides an antigenbinding site that recognizes and binds a conformational epitopepartially located in the V domain of the human CD33 extracellulardomain; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:270. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:270. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:316 as CDR1, SEQ ID NO:317 as CDR2, and SEQ IDNO:318 as CDR3 of SEQ ID NO:270. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:532 as CDR1,SEQ ID NO:317 as CDR2, and SEQ ID NO:533 as CDR3 of SEQ ID NO:270. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:270 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:270 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:271. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:270 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:271, which includes amino acid sequences SEQ ID NO:319 as CDR1, SEQID NO:320 as CDR2, and SEQ ID NO:321 as CDR3 of SEQ ID NO:271.

In certain embodiments, the present invention provides an antigenbinding site that recognizes and binds a conformational epitopepartially located in the V domain of the human CD33 extracellulardomain; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:272. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:272. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:322 as CDR1, SEQ ID NO:323 as CDR2, and SEQ IDNO:324 as CDR3 of SEQ ID NO:272. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:534 as CDR1,SEQ ID NO:323 as CDR2, and SEQ ID NO:535 as CDR3 of SEQ ID NO:272. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:272 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:272 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:273. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:272 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:273, which includes amino acid sequences SEQ ID NO:325 as CDR1, SEQID NO:326 as CDR2, and SEQ ID NO:327 as CDR3 of SEQ ID NO:273.

In certain embodiments, the present invention provides an antigenbinding site that recognizes and binds a conformational epitopepartially located in the V domain of the human CD33 extracellulardomain; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:280. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:280. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:346 as CDR1, SEQ ID NO:347 as CDR2, and SEQ IDNO:348 as CDR3 of SEQ ID NO:280. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:542 as CDR1,SEQ ID NO:347 as CDR2, and SEQ ID NO:543 as CDR3 of SEQ ID NO:280. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:280 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:280 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:281. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:280 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:281, which includes amino acid sequences SEQ ID NO:349 as CDR1, SEQID NO:350 as CDR2, and SEQ ID NO:351 as CDR3 of SEQ ID NO:281.

An Antigen Binding Site that Recognizes and Binds a ConformationalEpitope on an Extracellular Domain of the Human CD33 but not aConformational Epitope on an Extracellular Domain of theCynomolgus/Rhesus (Cyno) CD33

In one aspect, the present invention provides an antigen binding sitethat recognizes and binds one or more conformational epitopes on theextracellular domain of the human CD33 but does not recognize and/orbind one or more conformational epitopes on the extracellular domain ofthe cyno CD33.

In certain embodiments, the present invention provides an antigenbinding site that recognizes and binds one or more conformationalepitopes on the extracellular domain of the human CD33 but does notrecognize and/or bind one or more conformational epitopes on theextracellular domain of the cyno CD33; the antigen binding site includesa heavy chain variable domain including an amino acid sequence at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLEWMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYF DLWGRGTLVTVSS[SEQ ID NO:7]. In some embodiments, the antibody heavy chain variabledomain is at least 95% identical to SEQ ID NO:7. In some embodiments,the heavy chain variable domain incorporates amino acid sequencesYTFSDYYMH [SEQ ID NO:39] as CDR1, MINPSWGSTSYAQKFQG [SEQ ID NO:40] asCDR2, and AREAADGFVGERYFDL [SEQ ID NO:41] as CDR3 of SEQ ID NO:7. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:7 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:7 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVALPITFGGGTKVEIK [SEQ ID NO:8]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:7 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:8, which includes amino acid sequencesRSSQSLLYSNGYNYLD [SEQ ID NO:42] as CDR1, LGSNRAS [SEQ ID NO:43] as CDR2,and MQDVALPIT [SEQ ID NO:44] as CDR3 of SEQ ID NO:8.

In certain embodiments, the present invention provides an antigenbinding site that recognizes and binds one or more conformationalepitopes on the extracellular domain of the human CD33 but does notrecognize and/or bind one or more conformational epitopes on theextracellular domain of the cyno CD33; the antigen binding site includesa heavy chain variable domain including an amino acid sequence at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQGLEWMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDV WGKGTTVTVSS [SEQID NO:13]. In some embodiments, the antibody heavy chain variable domainis at least 95% identical to SEQ ID NO:13. In some embodiments, theheavy chain variable domain incorporates amino acid sequences YTFGTYYMH[SEQ ID NO:57] as CDR1, IINPSRGSTVYAQKFQG [SEQ ID NO:58] as CDR2, andARGAGYDDEDMDV [SEQ ID NO:59] as CDR3 of SEQ ID NO:13. In certainembodiments, the antibody heavy chain variable domain which includes anamino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:13 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:13 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFGGGTKVEIK [SEQ ID NO:14]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:13 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:14, which includes amino acid sequencesRASQGIDSWLA [SEQ ID NO:60] as CDR1, AASSLQS [SEQ ID NO:61] as CDR2, andQQAHSYPLT [SEQ ID NO:62] as CDR3 of SEQ ID NO:14.

In certain embodiments, an antibody binding site that includes a heavychain variable domain including an amino acid sequence at least 90%(e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQGLEWMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDV WGKGTTVTVSS [SEQID NO:13] binds to the full-length extracellular domain of human CD33,but does not bind human CD33 V domain or C domain individually, and doesnot cross-block binding to human CD33 with lintuzumab. In certainembodiments, an antibody binding site that includes a heavy chainvariable domain including an amino acid sequence at least 90% (e.g.,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:13, and is paired with an antibodylight chain variable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFGGGTKVEIK [SEQ ID NO:14], bindsto the full-length extracellular domain of human CD33, but does not bindhuman CD33 V domain or C domain individually, and does not cross-blockbinding to human CD33 with lintuzumab. In certain embodiments, anantibody binding site that includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:13, and is paired with an antibody light chain variabledomain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:14, whichincludes amino acid sequences RASQGIDSWLA [SEQ ID NO:60] as CDR1,AASSLQS [SEQ ID NO:61] as CDR2, and QQAHSYPLT [SEQ ID NO:62] as CDR3 ofSEQ ID NO:14, binds to the full-length extracellular domain of humanCD33, but does not bind human CD33 V domain or C domain individually,and does not cross-block binding to human CD33 with lintuzumab.

An Antigen Binding Site that Binds to the R69G Allele of Human CD33

In one aspect, the present invention provides an antigen binding sitethat binds to the R69G allele of human CD33. In certain embodiments, thepresent invention provides an antigen binding site that binds to theR69G allele of human CD33; the antigen binding site includes a heavychain variable domain including an amino acid sequence at least 90%(e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS [SEQ ID NO:1]. In some embodiments, the antibody heavychain variable domain is at least 95% identical to SEQ ID NO:1. In someembodiments, the heavy chain variable domain includes amino acidsequences FTFSSYGMS [SEQ ID NO:21] as the firstcomplementarity-determining region 1 (“CDR1”), NIKQDGSEKYYVDSVKG [SEQ IDNO:22] as the second CDR (“CDR2”), and AREGGPYYDSSGYFVYYGMDV [SEQ IDNO:23] as the third CDR (“CDR3”) of SEQ ID NO:1. In certain embodiments,the antibody heavy chain variable domain which includes an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence of SEQ ID NO:1 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:1can be paired with an antibody light chain variable domain at least 90%(e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESFPTFGGGTKVEIK [SEQ ID NO:2]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:1 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:2, which includes amino acid sequences RASQSISSWLA[SEQ ID NO:24] as CDR1, DASSLES [SEQ ID NO:25] as CDR2, and QQYESFPT[SEQ ID NO:26] as CDR3 of SEQ ID NO:2.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQ GTMVTVSS [SEQ IDNO:3]. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:3. In some embodiments, the heavychain variable domain incorporates amino acid sequences FTFSSYWMS [SEQID NO:27] as CDR1, NIKQDGSEKYYVDSVKG [SEQ ID NO:28] as CDR2, andARPLNAGELDV [SEQ ID NO:29] as CDR3 of SEQ ID NO:3. In certainembodiments, the antibody heavy chain variable domain which includes anamino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:3 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:3 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLESYPLTFGGGTKVEIK [SEQ ID NO:4]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:3 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:4, which includes amino acid sequences RASQSISSWLA[SEQ ID NO:30] as CDR1, EASSLES [SEQ ID NO:31] as CDR2, and QQLESYPLT[SEQ ID NO:32] as CDR3 of SEQ ID NO:4.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMSWVRQAPGKGLEWVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS [SEQ ID NO:5]. In some embodiments, the antibody heavychain variable domain is at least 95% identical to SEQ ID NO:5. In someembodiments, the heavy chain variable domain incorporates amino acidsequences FTFSKYTMS [SEQ ID NO:33] as CDR1, AIVGSGESTYFADSVKG [SEQ IDNO:34] as CDR2, and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO:35] as CDR3 of SEQID NO:5. In some embodiments, the heavy chain variable domainincorporates amino acid sequences KYTMS [SEQ ID NO:183] as CDR1,AIVGSGESTYFADSVKG [SEQ ID NO:34] as CDR2, and EGGPYYDSSGYFVYYGMDV [SEQID NO:184] as CDR3 of SEQ ID NO:5. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:5 is combined with alight chain variable domain to form an antigen-binding site capable ofbinding to CD33. For example, an antibody heavy chain variable domain atleast 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:5 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK [SEQ ID NO:6]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:5 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:6, which includes amino acid sequences RASQSISSWLA[SEQ ID NO:36] as CDR1, KASSLES [SEQ ID NO:37] or KASSLE [SEQ ID NO:185]as CDR2, and QQYDDLPT [SEQ ID NO:38] as CDR3 of SEQ ID NO:6.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKGLEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQ GTMVTVSS [SEQ IDNO:9]. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:9. In some embodiments, the heavychain variable domain incorporates amino acid sequences FTFGSYWMS [SEQID NO:45] as CDR1, TIKQDGSEKSYVDSVKG [SEQ ID NO:46] as CDR2, andARPLNAGELDV [SEQ ID NO:47] as CDR3 of SEQ ID NO:9. In some embodiments,the heavy chain variable domain incorporates amino acid sequences SYWMS[SEQ ID NO:181] as CDR1, TIKQDGSEKSYVDSVKG [SEQ ID NO:46] as CDR2, andRPLNAGELDV [SEQ ID NO:182] as CDR3 of SEQ ID NO:9. In certainembodiments, the antibody heavy chain variable domain which includes anamino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:9 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:9 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGGGTKVEIK [SEQ ID NO:10]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:9 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:10, which includes amino acid sequencesRASQSISSWLA [SEQ ID NO:48] as CDR1, EASSLES [SEQ ID NO:49] as CDR2, andQQSQSYPPIT [SEQ ID NO:50] as CDR3 of SEQ ID NO:10.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGKGLEWVATIKRDGSEKGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQG TMVTVSS [SEQ IDNO:11]. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:11. In some embodiments, the heavychain variable domain incorporates amino acid sequences FTFPSYWMS [SEQID NO:51] as CDR1, TIKRDGSEKGYVDSVKG [SEQ ID NO:52] as CDR2, andARPLNAGELDV [SEQ ID NO:53] as CDR3 of SEQ ID NO:11. In certainembodiments, the antibody heavy chain variable domain which includes anamino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:11 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:11 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGGGTKVEIK [SEQ ID NO:12]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:11 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:12, which includes amino acid sequencesRASQSISSWLA [SEQ ID NO:54] as CDR1, EASSLES [SEQ ID NO:55] as CDR2, andQQSQSYPPIT [SEQ ID NO:56] as CDR3 of SEQ ID NO:12.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQGLEWMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDV WGKGTTVTVSS [SEQID NO:13]. In some embodiments, the antibody heavy chain variable domainis at least 95% identical to SEQ ID NO:13. In some embodiments, theheavy chain variable domain incorporates amino acid sequences YTFGTYYMH[SEQ ID NO:57] as CDR1, IINPSRGSTVYAQKFQG [SEQ ID NO:58] as CDR2, andARGAGYDDEDMDV [SEQ ID NO:59] as CDR3 of SEQ ID NO:13. In certainembodiments, the antibody heavy chain variable domain which includes anamino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:13 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:13 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFGGGTKVEIK [SEQ ID NO:14]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:13 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:14, which includes amino acid sequencesRASQGIDSWLA [SEQ ID NO:60] as CDR1, AASSLQS [SEQ ID NO:61] as CDR2, andQQAHSYPLT [SEQ ID NO:62] as CDR3 of SEQ ID NO:14.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence ofEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSSSEGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYY GMDVWGQGTTVTVSS[SEQ ID NO:15]. In some embodiments, the antibody heavy chain variabledomain is at least 95% identical to SEQ ID NO:15. In some embodiments,the heavy chain variable domain incorporates amino acid sequencesFTFSSYAMS [SEQ ID NO:63] as CDR1, SISSSSEGIYYADSVKG [SEQ ID NO:64] asCDR2, and AREGGPYYDSSGYFVYYGMDV [SEQ ID NO:65] as CDR3 of SEQ ID NO:15.In certain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:15 is combined with a light chain variable domain to forman antigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:15 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK [SEQ ID NO:16]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:15 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:16, which includes amino acid sequencesRASNSISSWLA [SEQ ID NO:66] as CDR1, EASSTKS [SEQ ID NO:67] as CDR2, andQQYDDLPT [SEQ ID NO:68] as CDR3 of SEQ ID NO:16.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINTDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFD YWGQGTLVTVSS[SEQ ID NO:17]. In some embodiments, the antibody heavy chain variabledomain is at least 95% identical to SEQ ID NO:17. In some embodiments,the heavy chain variable domain incorporates amino acid sequencesFTFSSYWMS [SEQ ID NO:69] as CDR1, NINTDGSEVYYVDSVKG [SEQ ID NO:70] asCDR2, and ARDVGPGIAYQGHFDY [SEQ ID NO:71] as CDR3 of SEQ ID NO:17. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:17 is combined with a light chain variable domain to forman antigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:17 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQVIYSYLNWYQQKPGKAPKLLIYAASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPLTFGGGTKVEIK [SEQ ID NO:18]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:17 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:18, which includes amino acid sequencesRASQVIYSYLN [SEQ ID NO:72] as CDR1, AASSLKS [SEQ ID NO:73] as CDR2, andQQVYDTPLT [SEQ ID NO:74] as CDR3 of SEQ ID NO:18.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence ofQLQLQESGPGLVKPSETLSLTCTVSGGSISSTDYYWGWIRQPPGKGLEWIGSIGYSGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQG TTVTVSS [SEQ IDNO:19]. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:19. In some embodiments, the heavychain variable domain incorporates amino acid sequences GSISSTDYYWG [SEQID NO:75] as CDR1, SIGYSGTYYNPSLKS [SEQ ID NO:76] as CDR2, andARETAHDVHGMDV [SEQ ID NO:77] as CDR3 of SEQ ID NO:19. In certainembodiments, the antibody heavy chain variable domain which includes anamino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:19 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:19 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceEIVLTQSPATLSLSPGERATLSCRASHSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPTFGGGTKVEIK [SEQ ID NO:20]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:19 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:20, which includes amino acid sequencesRASHSVYSYLA [SEQ ID NO:78] as CDR1, DASNRAT [SEQ ID NO:79] as CDR2, andQQYDNLPT [SEQ ID NO:80] as CDR3 of SEQ ID NO:20.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:266. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:266. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:304 asCDR1, SEQ ID NO:305 as CDR2, and SEQ ID NO:306 as CDR3 of SEQ ID NO:266.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:528 as CDR1, SEQ ID NO:305 as CDR2, and SEQ IDNO:529 as CDR3 of SEQ ID NO:266. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:266 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:266 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:267. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:266 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:267, which includes amino acidsequences SEQ ID NO:307 as CDR1, SEQ ID NO:308 as CDR2, and SEQ IDNO:309 as CDR3 of SEQ ID NO:267.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:268. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:268. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:310 asCDR1, SEQ ID NO:311 as CDR2, and SEQ ID NO:312 as CDR3 of SEQ ID NO:268.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:530 as CDR1, SEQ ID NO:311 as CDR2, and SEQ IDNO:531 as CDR3 of SEQ ID NO:268. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:268 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:268 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:269. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:268 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:269, which includes amino acidsequences SEQ ID NO:313 as CDR1, SEQ ID NO:314 as CDR2, and SEQ IDNO:315 as CDR3 of SEQ ID NO:269.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:270. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:270. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:316 asCDR1, SEQ ID NO:317 as CDR2, and SEQ ID NO:318 as CDR3 of SEQ ID NO:270.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:532 as CDR1, SEQ ID NO:317 as CDR2, and SEQ IDNO:533 as CDR3 of SEQ ID NO:270. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:270 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:270 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:271. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:270 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:271, which includes amino acidsequences SEQ ID NO:319 as CDR1, SEQ ID NO:320 as CDR2, and SEQ IDNO:321 as CDR3 of SEQ ID NO:271.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:272. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:272. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:322 asCDR1, SEQ ID NO:323 as CDR2, and SEQ ID NO:324 as CDR3 of SEQ ID NO:272.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:534 as CDR1, SEQ ID NO:323 as CDR2, and SEQ IDNO:535 as CDR3 of SEQ ID NO:272. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:272 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:272 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:273. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:272 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:273, which includes amino acidsequences SEQ ID NO:325 as CDR1, SEQ ID NO:326 as CDR2, and SEQ IDNO:327 as CDR3 of SEQ ID NO:273.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:274. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:274. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:328 asCDR1, SEQ ID NO:329 as CDR2, and SEQ ID NO:330 as CDR3 of SEQ ID NO:274.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:536 as CDR1, SEQ ID NO:329 as CDR2, and SEQ IDNO:537 as CDR3 of SEQ ID NO:274. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:274 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:274 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:275. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:274 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:275, which includes amino acidsequences SEQ ID NO:331 as CDR1, SEQ ID NO:332 as CDR2, and SEQ IDNO:333 as CDR3 of SEQ ID NO:275.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:276. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:276. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:334 asCDR1, SEQ ID NO:335 as CDR2, and SEQ ID NO:336 as CDR3 of SEQ ID NO:276.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:538 as CDR1, SEQ ID NO:335 as CDR2, and SEQ IDNO:539 as CDR3 of SEQ ID NO:276. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:276 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:276 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:277. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:276 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:277, which includes amino acidsequences SEQ ID NO:337 as CDR1, SEQ ID NO:338 as CDR2, and SEQ IDNO:339 as CDR3 of SEQ ID NO:277.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:278. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:278. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:340 asCDR1, SEQ ID NO:341 as CDR2, and SEQ ID NO:342 as CDR3 of SEQ ID NO:278.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:540 as CDR1, SEQ ID NO:341 as CDR2, and SEQ IDNO:541 as CDR3 of SEQ ID NO:278. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:278 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:278 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:279. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:278 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:279, which includes amino acidsequences SEQ ID NO:343 as CDR1, SEQ ID NO:344 as CDR2, and SEQ IDNO:345 as CDR3 of SEQ ID NO:279.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:280. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:280. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:346 asCDR1, SEQ ID NO:347 as CDR2, and SEQ ID NO:348 as CDR3 of SEQ ID NO:280.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:542 as CDR1, SEQ ID NO:347 as CDR2, and SEQ IDNO:543 as CDR3 of SEQ ID NO:280. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:280 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:280 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:281. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:280 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:281, which includes amino acidsequences SEQ ID NO:349 as CDR1, SEQ ID NO:350 as CDR2, and SEQ IDNO:351 as CDR3 of SEQ ID NO:281.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:282. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:282. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:352 asCDR1, SEQ ID NO:353 as CDR2, and SEQ ID NO:354 as CDR3 of SEQ ID NO:282.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:544 as CDR1, SEQ ID NO:353 as CDR2, and SEQ IDNO:545 as CDR3 of SEQ ID NO:282. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:282 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:282 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:283. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:282 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:283, which includes amino acidsequences SEQ ID NO:355 as CDR1, SEQ ID NO:356 as CDR2, and SEQ IDNO:357 as CDR3 of SEQ ID NO:283.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:284. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:284. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:358 asCDR1, SEQ ID NO:359 as CDR2, and SEQ ID NO:360 as CDR3 of SEQ ID NO:284.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:546 as CDR1, SEQ ID NO:359 as CDR2, and SEQ IDNO:360 as CDR3 of SEQ ID NO:547. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:284 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:284 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:285. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:284 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:285, which includes amino acidsequences SEQ ID NO:361 as CDR1, SEQ ID NO:362 as CDR2, and SEQ IDNO:363 as CDR3 of SEQ ID NO:285.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:286. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:286. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:364 asCDR1, SEQ ID NO:365 as CDR2, and SEQ ID NO:366 as CDR3 of SEQ ID NO:286.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:548 as CDR1, SEQ ID NO:365 as CDR2, and SEQ IDNO:549 as CDR3 of SEQ ID NO:286. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:286 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:286 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:287. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:286 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:287, which includes amino acidsequences SEQ ID NO:367 as CDR1, SEQ ID NO:368 as CDR2, and SEQ IDNO:369 as CDR3 of SEQ ID NO:287.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:292. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:292. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:382 asCDR1, SEQ ID NO:383 as CDR2, and SEQ ID NO:384 as CDR3 of SEQ ID NO:292.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:554 as CDR1, SEQ ID NO:383 as CDR2, and SEQ IDNO:555 as CDR3 of SEQ ID NO:292. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:292 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:292 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:293. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:292 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:293, which includes amino acidsequences SEQ ID NO:385 as CDR1, SEQ ID NO:386 as CDR2, and SEQ IDNO:387 as CDR3 of SEQ ID NO:293.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:294. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:294. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:388 asCDR1, SEQ ID NO:389 as CDR2, and SEQ ID NO:390 as CDR3 of SEQ ID NO:294.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:556 as CDR1, SEQ ID NO:389 as CDR2, and SEQ IDNO:557 as CDR3 of SEQ ID NO:294. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:294 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:294 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:295. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:294 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:295, which includes amino acidsequences SEQ ID NO:391 as CDR1, SEQ ID NO:392 as CDR2, and SEQ IDNO:393 as CDR3 of SEQ ID NO:295.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:296. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:296. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:394 asCDR1, SEQ ID NO:395 as CDR2, and SEQ ID NO:396 as CDR3 of SEQ ID NO:296.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:558 as CDR1, SEQ ID NO:395 as CDR2, and SEQ IDNO:559 as CDR3 of SEQ ID NO:296. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:296 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:296 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:297. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:296 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:297, which includes amino acidsequences SEQ ID NO:397 as CDR1, SEQ ID NO:398 as CDR2, and SEQ IDNO:399 as CDR3 of SEQ ID NO:297.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:298. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:298. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:400 asCDR1, SEQ ID NO:401 as CDR2, and SEQ ID NO:402 as CDR3 of SEQ ID NO:298.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:560 as CDR1, SEQ ID NO:401 as CDR2, and SEQ IDNO:561 as CDR3 of SEQ ID NO:298. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:298 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:298 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:299. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:298 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:299, which includes amino acidsequences SEQ ID NO:403 as CDR1, SEQ ID NO:404 as CDR2, and SEQ IDNO:405 as CDR3 of SEQ ID NO:299.

In certain embodiments, the present invention provides an antigenbinding site that binds to the R69G allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:302. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:302. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:412 asCDR1, SEQ ID NO:413 as CDR2, and SEQ ID NO:414 as CDR3 of SEQ ID NO:302.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:564 as CDR1, SEQ ID NO:413 as CDR2, and SEQ IDNO:565 as CDR3 of SEQ ID NO:302. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:302 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:302 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:303. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:302 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:303, which includes amino acidsequences SEQ ID NO:415 as CDR1, SEQ ID NO:416 as CDR2, and SEQ IDNO:417 as CDR3 of SEQ ID NO:303.

An antigen binding site that does not bind to the R69G allele of humanCD33

In one aspect, the present invention provides an antigen binding siteincluding a heavy chain variable domain that binds wild-type human CD33,but not the R69G allele of human CD33. In certain embodiments, thepresent invention provides an antigen binding site that does not bind tothe R69G allele of human CD33; the antigen binding site includes a heavychain variable domain including an amino acid sequence at least 90%(e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLEWMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYF DLWGRGTLVTVSS[SEQ ID NO:7]. In some embodiments, the antibody heavy chain variabledomain is at least 95% identical to SEQ ID NO:7. In some embodiments,the heavy chain variable domain incorporates amino acid sequencesYTFSDYYMH [SEQ ID NO:39] as CDR1, MINPSWGSTSYAQKFQG [SEQ ID NO:40] asCDR2, and AREAADGFVGERYFDL [SEQ ID NO:41] as CDR3 of SEQ ID NO:7. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:7 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:7 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVALPITFGGGTKVEIK [SEQ ID NO:8]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:7 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:8, which includes amino acid sequencesRSSQSLLYSNGYNYLD [SEQ ID NO:42] as CDR1, LGSNRAS [SEQ ID NO:43] as CDR2,and MQDVALPIT [SEQ ID NO:44] as CDR3 of SEQ ID NO:8.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the R69G alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:288. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:288.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:370 as CDR1, SEQ ID NO:371 as CDR2, and SEQ IDNO:372 as CDR3 of SEQ ID NO:288. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:550 as CDR1,SEQ ID NO:371 as CDR2, and SEQ ID NO:551 as CDR3 of SEQ ID NO:288. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:288 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:288 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:289. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:288 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:289, which includes amino acid sequences SEQ ID NO:373 as CDR1, SEQID NO:374 as CDR2, and SEQ ID NO:375 as CDR3 of SEQ ID NO:289.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the R69G alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:290. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:290.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:376 as CDR1, SEQ ID NO:377 as CDR2, and SEQ IDNO:378 as CDR3 of SEQ ID NO:290. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:552 as CDR1,SEQ ID NO:377 as CDR2, and SEQ ID NO:553 as CDR3 of SEQ ID NO:290. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:290 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:290 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:291. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:290 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:291, which includes amino acid sequences SEQ ID NO:379 as CDR1, SEQID NO:380 as CDR2, and SEQ ID NO:381 as CDR3 of SEQ ID NO:291.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the R69G alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:300. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:300.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:406 as CDR1, SEQ ID NO:407 as CDR2, and SEQ IDNO:408 as CDR3 of SEQ ID NO:300. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:562 as CDR1,SEQ ID NO:407 as CDR2, and SEQ ID NO:563 as CDR3 of SEQ ID NO:300. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:300 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:300 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:301. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:300 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:301, which includes amino acid sequences SEQ ID NO:409 as CDR1, SEQID NO:410 as CDR2, and SEQ ID NO:411 as CDR3 of SEQ ID NO:301.

An Antigen Binding Site that Binds to a Unique Epitope Including R69 onHuman CD33

In one aspect, the present invention provides an antigen binding siteincluding a heavy chain variable domain that binds to a unique epitopeon human CD33 that includes R69. In certain embodiments, the presentinvention provides an antigen binding site that binds to a uniqueepitope on human CD33 that includes R69; the antigen binding siteincludes a heavy chain variable domain including an amino acid sequenceat least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence ofQVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQGLEWMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYF DLWGRGTLVTVSS[SEQ ID NO:7].

In some embodiments, the antibody heavy chain variable domain is atleast 95% identical to SEQ ID NO:7. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences YTFSDYYMH [SEQ IDNO:39] as CDR1, MINPSWGSTSYAQKFQG [SEQ ID NO:40] as CDR2, andAREAADGFVGERYFDL [SEQ ID NO:41] as CDR3 of SEQ ID NO:7. In certainembodiments, the antibody heavy chain variable domain which includes anamino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:7 is combined with a light chain variable domain to form anantigen-binding site capable of binding to CD33. For example, anantibody heavy chain variable domain at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:7 can be paired with an antibody light chainvariable domain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVALPITFGGGTKVEIK [SEQ ID NO:8]. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:7 can be paired withan antibody light chain variable domain at least 90% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:8, which includes amino acid sequencesRSSQSLLYSNGYNYLD [SEQ ID NO:42] as CDR1, LGSNRAS [SEQ ID NO:43] as CDR2,and MQDVALPIT [SEQ ID NO:44] as CDR3 of SEQ ID NO:8.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesR69; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:288. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:288. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:370 as CDR1, SEQ ID NO:371 as CDR2, and SEQ IDNO:372 as CDR3 of SEQ ID NO:288. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:550 as CDR1,SEQ ID NO:371 as CDR2, and SEQ ID NO:551 as CDR3 of SEQ ID NO:288. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:288 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:288 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:289. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:288 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:289, which includes amino acid sequences SEQ ID NO:373 as CDR1, SEQID NO:374 as CDR2, and SEQ ID NO:375 as CDR3 of SEQ ID NO:289.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesR69; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:290. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:290. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:376 as CDR1, SEQ ID NO:377 as CDR2, and SEQ IDNO:378 as CDR3 of SEQ ID NO:290. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:552 as CDR1,SEQ ID NO:377 as CDR2, and SEQ ID NO:553 as CDR3 of SEQ ID NO:290. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:290 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:290 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:291. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:290 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:291, which includes amino acid sequences SEQ ID NO:379 as CDR1, SEQID NO:380 as CDR2, and SEQ ID NO:381 as CDR3 of SEQ ID NO:291.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesR69; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:300. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:300. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:406 as CDR1, SEQ ID NO:407 as CDR2, and SEQ IDNO:408 as CDR3 of SEQ ID NO:300. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:562 as CDR1,SEQ ID NO:407 as CDR2, and SEQ ID NO:563 as CDR3 of SEQ ID NO:300. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:300 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:300 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:301. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:300 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:301, which includes amino acid sequences SEQ ID NO:409 as CDR1, SEQID NO:410 as CDR2, and SEQ ID NO:411 as CDR3 of SEQ ID NO:301.

An Antigen Binding Site that Binds to the S128N Allele of Human CD33

In one aspect, the present invention provides an antigen binding sitethat binds to the S128N allele of human CD33.

In certain embodiments, the present invention provides an antigenbinding site that binds to the S128N allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:268. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:268. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:310 asCDR1, SEQ ID NO:311 as CDR2, and SEQ ID NO:312 as CDR3 of SEQ ID NO:268.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:530 as CDR1, SEQ ID NO:311 as CDR2, and SEQ IDNO:531 as CDR3 of SEQ ID NO:268. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:268 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:268 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:269. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:268 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:269, which includes amino acidsequences SEQ ID NO:313 as CDR1, SEQ ID NO:314 as CDR2, and SEQ IDNO:315 as CDR3 of SEQ ID NO:269.

In certain embodiments, the present invention provides an antigenbinding site that binds to the S128N allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:274. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:274. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:328 asCDR1, SEQ ID NO:329 as CDR2, and SEQ ID NO:330 as CDR3 of SEQ ID NO:274.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:536 as CDR1, SEQ ID NO:329 as CDR2, and SEQ IDNO:537 as CDR3 of SEQ ID NO:274. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:274 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:274 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:275. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:274 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:275, which includes amino acidsequences SEQ ID NO:331 as CDR1, SEQ ID NO:332 as CDR2, and SEQ IDNO:333 as CDR3 of SEQ ID NO:275.

In certain embodiments, the present invention provides an antigenbinding site that binds to the S128N allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:276. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:276. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:334 asCDR1, SEQ ID NO:335 as CDR2, and SEQ ID NO:336 as CDR3 of SEQ ID NO:276.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:538 as CDR1, SEQ ID NO:335 as CDR2, and SEQ IDNO:539 as CDR3 of SEQ ID NO:276. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:276 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:276 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:277. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:276 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:277, which includes amino acidsequences SEQ ID NO:337 as CDR1, SEQ ID NO:338 as CDR2, and SEQ IDNO:339 as CDR3 of SEQ ID NO:277.

In certain embodiments, the present invention provides an antigenbinding site that binds to the S128N allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:292. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:292. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:382 asCDR1, SEQ ID NO:383 as CDR2, and SEQ ID NO:384 as CDR3 of SEQ ID NO:292.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:554 as CDR1, SEQ ID NO:383 as CDR2, and SEQ IDNO:555 as CDR3 of SEQ ID NO:292. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:292 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:292 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:293. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:292 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:293, which includes amino acidsequences SEQ ID NO:385 as CDR1, SEQ ID NO:386 as CDR2, and SEQ IDNO:387 as CDR3 of SEQ ID NO:293.

In certain embodiments, the present invention provides an antigenbinding site that binds to the S128N allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:294. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:294. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:388 asCDR1, SEQ ID NO:389 as CDR2, and SEQ ID NO:390 as CDR3 of SEQ ID NO:294.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:556 as CDR1, SEQ ID NO:389 as CDR2, and SEQ IDNO:557 as CDR3 of SEQ ID NO:294. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:294 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:294 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:295. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:294 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:295, which includes amino acidsequences SEQ ID NO:391 as CDR1, SEQ ID NO:392 as CDR2, and SEQ IDNO:393 as CDR3 of SEQ ID NO:295.

In certain embodiments, the present invention provides an antigenbinding site that binds to the S128N allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:296. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:296. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:394 asCDR1, SEQ ID NO:395 as CDR2, and SEQ ID NO:396 as CDR3 of SEQ ID NO:296.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:558 as CDR1, SEQ ID NO:395 as CDR2, and SEQ IDNO:559 as CDR3 of SEQ ID NO:296. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:296 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:296 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:297. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:296 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:297, which includes amino acidsequences SEQ ID NO:397 as CDR1, SEQ ID NO:398 as CDR2, and SEQ IDNO:399 as CDR3 of SEQ ID NO:297.

In certain embodiments, the present invention provides an antigenbinding site that binds to the S128N allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:298. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:298. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:400 asCDR1, SEQ ID NO:401 as CDR2, and SEQ ID NO:402 as CDR3 of SEQ ID NO:298.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:560 as CDR1, SEQ ID NO:401 as CDR2, and SEQ IDNO:561 as CDR3 of SEQ ID NO:298. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:298 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:298 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:299. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:298 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:299, which includes amino acidsequences SEQ ID NO:403 as CDR1, SEQ ID NO:404 as CDR2, and SEQ IDNO:405 as CDR3 of SEQ ID NO:299.

In certain embodiments, the present invention provides an antigenbinding site that binds to the S128N allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:290. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:290. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:376 asCDR1, SEQ ID NO:377 as CDR2, and SEQ ID NO:378 as CDR3 of SEQ ID NO:290.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:552 as CDR1, SEQ ID NO:377 as CDR2, and SEQ IDNO:553 as CDR3 of SEQ ID NO:290. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:290 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:290 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:291. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:290 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:291, which includes amino acidsequences SEQ ID NO:379 as CDR1, SEQ ID NO:380 as CDR2, and SEQ IDNO:381 as CDR3 of SEQ ID NO:291.

In certain embodiments, the present invention provides an antigenbinding site that binds to the S128N allele of human CD33; the antigenbinding site includes a heavy chain variable domain including an aminoacid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:300. In some embodiments, the antibody heavy chain variable domain isat least 95% identical to SEQ ID NO:300. In some embodiments, the heavychain variable domain incorporates amino acid sequences SEQ ID NO:406 asCDR1, SEQ ID NO:407 as CDR2, and SEQ ID NO:408 as CDR3 of SEQ ID NO:300.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:562 as CDR1, SEQ ID NO:407 as CDR2, and SEQ IDNO:563 as CDR3 of SEQ ID NO:300. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:300 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:300 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:301. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:300 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:301, which includes amino acidsequences SEQ ID NO:409 as CDR1, SEQ ID NO:410 as CDR2, and SEQ IDNO:411 as CDR3 of SEQ ID NO:301.

An Antigen Binding Site that does not Bind to the S128N Allele of HumanCD33

In one aspect, the present invention provides an antigen binding siteincluding a heavy chain variable domain that binds to wild-type humanCD33 but not the S128N allele of human CD33.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the S128N alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:302. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:302.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:412 as CDR1, SEQ ID NO:413 as CDR2, and SEQ IDNO:414 as CDR3 of SEQ ID NO:302. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:564 as CDR1,SEQ ID NO:413 as CDR2, and SEQ ID NO:565 as CDR3 of SEQ ID NO:302. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:302 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:302 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:303. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:302 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:303, which includes amino acid sequences SEQ ID NO:415 as CDR1, SEQID NO:416 as CDR2, and SEQ ID NO:417 as CDR3 of SEQ ID NO:303.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the S128N alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:266. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:266.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:304 as CDR1, SEQ ID NO:305 as CDR2, and SEQ IDNO:306 as CDR3 of SEQ ID NO:266. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:528 as CDR1,SEQ ID NO:305 as CDR2, and SEQ ID NO:529 as CDR3 of SEQ ID NO:266. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:266 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:266 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:267. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:266 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:267, which includes amino acid sequences SEQ ID NO:307 as CDR1, SEQID NO:308 as CDR2, and SEQ ID NO:309 as CDR3 of SEQ ID NO:267.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the S128N alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:270. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:270.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:316 as CDR1, SEQ ID NO:317 as CDR2, and SEQ IDNO:318 as CDR3 of SEQ ID NO:270. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:532 as CDR1,SEQ ID NO:317 as CDR2, and SEQ ID NO:533 as CDR3 of SEQ ID NO:270. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:270 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:270 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:271. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:270 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:271, which includes amino acid sequences SEQ ID NO:319 as CDR1, SEQID NO:320 as CDR2, and SEQ ID NO:321 as CDR3 of SEQ ID NO:271.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the S128N alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:272. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:272.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:322 as CDR1, SEQ ID NO:323 as CDR2, and SEQ IDNO:324 as CDR3 of SEQ ID NO:272. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:534 as CDR1,SEQ ID NO:323 as CDR2, and SEQ ID NO:535 as CDR3 of SEQ ID NO:272. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:272 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:272 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:273. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:272 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:273, which includes amino acid sequences SEQ ID NO:325 as CDR1, SEQID NO:326 as CDR2, and SEQ ID NO:327 as CDR3 of SEQ ID NO:273.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the S128N alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:278. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:278.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:340 as CDR1, SEQ ID NO:341 as CDR2, and SEQ IDNO:342 as CDR3 of SEQ ID NO:278. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:540 as CDR1,SEQ ID NO:341 as CDR2, and SEQ ID NO:541 as CDR3 of SEQ ID NO:278. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:278 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:278 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:279. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:278 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:279, which includes amino acid sequences SEQ ID NO:343 as CDR1, SEQID NO:344 as CDR2, and SEQ ID NO:345 as CDR3 of SEQ ID NO:279.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the S128N alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:280. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:280.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:346 as CDR1, SEQ ID NO:347 as CDR2, and SEQ IDNO:348 as CDR3 of SEQ ID NO:280. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:542 as CDR1,SEQ ID NO:347 as CDR2, and SEQ ID NO:543 as CDR3 of SEQ ID NO:280. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:280 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:280 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:281. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:280 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:281, which includes amino acid sequences SEQ ID NO:349 as CDR1, SEQID NO:350 as CDR2, and SEQ ID NO:351 as CDR3 of SEQ ID NO:281.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the S128N alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:282. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:282.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:352 as CDR1, SEQ ID NO:353 as CDR2, and SEQ IDNO:354 as CDR3 of SEQ ID NO:282. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:544 as CDR1,SEQ ID NO:353 as CDR2, and SEQ ID NO:545 as CDR3 of SEQ ID NO:282. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:282 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:282 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:283. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:282 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:283, which includes amino acid sequences SEQ ID NO:355 as CDR1, SEQID NO:356 as CDR2, and SEQ ID NO:357 as CDR3 of SEQ ID NO:283.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the S128N alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:284. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:284.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:358 as CDR1, SEQ ID NO:359 as CDR2, and SEQ IDNO:360 as CDR3 of SEQ ID NO:284. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:546 as CDR1,SEQ ID NO:359 as CDR2, and SEQ ID NO:360 as CDR3 of SEQ ID NO:547. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:284 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:284 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:285. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:284 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:285, which includes amino acid sequences SEQ ID NO:361 as CDR1, SEQID NO:362 as CDR2, and SEQ ID NO:363 as CDR3 of SEQ ID NO:285.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the S128N alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:286. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:286.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:364 as CDR1, SEQ ID NO:365 as CDR2, and SEQ IDNO:366 as CDR3 of SEQ ID NO:286. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:548 as CDR1,SEQ ID NO:365 as CDR2, and SEQ ID NO:549 as CDR3 of SEQ ID NO:286. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:286 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:286 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:287. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:286 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:287, which includes amino acid sequences SEQ ID NO:367 as CDR1, SEQID NO:368 as CDR2, and SEQ ID NO:369 as CDR3 of SEQ ID NO:287.

In certain embodiments, the present invention provides an antigenbinding site that binds to wild-type human CD33 but not the S128N alleleof human CD33; the antigen binding site includes a heavy chain variabledomain including an amino acid sequence at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:288. In some embodiments, the antibodyheavy chain variable domain is at least 95% identical to SEQ ID NO:288.In some embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:370 as CDR1, SEQ ID NO:371 as CDR2, and SEQ IDNO:372 as CDR3 of SEQ ID NO:288. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:550 as CDR1,SEQ ID NO:371 as CDR2, and SEQ ID NO:551 as CDR3 of SEQ ID NO:288. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:288 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:288 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:289. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:288 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:289, which includes amino acid sequences SEQ ID NO:373 as CDR1, SEQID NO:374 as CDR2, and SEQ ID NO:375 as CDR3 of SEQ ID NO:289.

An Antigen Binding Site that Binds to a Unique Epitope Including S128 onHuman CD33

In one aspect, the present invention provides an antigen binding siteincluding a heavy chain variable domain that binds to a unique epitopeon human CD33 that includes S128.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesS128; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:302. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:302. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:412 as CDR1, SEQ ID NO:413 as CDR2, and SEQ IDNO:414 as CDR3 of SEQ ID NO:302. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:564 as CDR1,SEQ ID NO:413 as CDR2, and SEQ ID NO:565 as CDR3 of SEQ ID NO:302. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:302 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:302 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:303. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:302 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:303, which includes amino acid sequences SEQ ID NO:415 as CDR1, SEQID NO:416 as CDR2, and SEQ ID NO:417 as CDR3 of SEQ ID NO:303.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesS128; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:266. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:266. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:304 as CDR1, SEQ ID NO:305 as CDR2, and SEQ IDNO:306 as CDR3 of SEQ ID NO:266. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:528 as CDR1,SEQ ID NO:305 as CDR2, and SEQ ID NO:529 as CDR3 of SEQ ID NO:266. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:266 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:266 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:267. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:266 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:267, which includes amino acid sequences SEQ ID NO:307 as CDR1, SEQID NO:308 as CDR2, and SEQ ID NO:309 as CDR3 of SEQ ID NO:267.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesS128; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:270. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:270. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:316 as CDR1, SEQ ID NO:317 as CDR2, and SEQ IDNO:318 as CDR3 of SEQ ID NO:270. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:532 as CDR1,SEQ ID NO:317 as CDR2, and SEQ ID NO:533 as CDR3 of SEQ ID NO:270. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:270 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:270 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:271. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:270 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:271, which includes amino acid sequences SEQ ID NO:319 as CDR1, SEQID NO:320 as CDR2, and SEQ ID NO:321 as CDR3 of SEQ ID NO:271.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesS128; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:272. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:272. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:322 as CDR1, SEQ ID NO:323 as CDR2, and SEQ IDNO:324 as CDR3 of SEQ ID NO:272. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:534 as CDR1,SEQ ID NO:323 as CDR2, and SEQ ID NO:535 as CDR3 of SEQ ID NO:272. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:272 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:272 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:273. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:272 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:273, which includes amino acid sequences SEQ ID NO:325 as CDR1, SEQID NO:326 as CDR2, and SEQ ID NO:327 as CDR3 of SEQ ID NO:273.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesS128; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:278. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:278. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:340 as CDR1, SEQ ID NO:341 as CDR2, and SEQ IDNO:342 as CDR3 of SEQ ID NO:278. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:540 as CDR1,SEQ ID NO:341 as CDR2, and SEQ ID NO:541 as CDR3 of SEQ ID NO:278. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:278 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:278 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:279. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:278 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:279, which includes amino acid sequences SEQ ID NO:343 as CDR1, SEQID NO:344 as CDR2, and SEQ ID NO:345 as CDR3 of SEQ ID NO:279.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesS128; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:280. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:280. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:346 as CDR1, SEQ ID NO:347 as CDR2, and SEQ IDNO:348 as CDR3 of SEQ ID NO:280. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:542 as CDR1,SEQ ID NO:347 as CDR2, and SEQ ID NO:543 as CDR3 of SEQ ID NO:280. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:280 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:280 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:281. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:280 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:281, which includes amino acid sequences SEQ ID NO:349 as CDR1, SEQID NO:350 as CDR2, and SEQ ID NO:351 as CDR3 of SEQ ID NO:281.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesS128; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:282. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:282. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:352 as CDR1, SEQ ID NO:353 as CDR2, and SEQ IDNO:354 as CDR3 of SEQ ID NO:282. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:544 as CDR1,SEQ ID NO:353 as CDR2, and SEQ ID NO:545 as CDR3 of SEQ ID NO:282. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:282 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:282 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:283. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:282 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:283, which includes amino acid sequences SEQ ID NO:355 as CDR1, SEQID NO:356 as CDR2, and SEQ ID NO:357 as CDR3 of SEQ ID NO:283.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesS128; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:284. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:284. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:358 as CDR1, SEQ ID NO:359 as CDR2, and SEQ IDNO:360 as CDR3 of SEQ ID NO:284. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:546 as CDR1,SEQ ID NO:359 as CDR2, and SEQ ID NO:360 as CDR3 of SEQ ID NO:547. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:284 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:284 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:285. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:284 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:285, which includes amino acid sequences SEQ ID NO:361 as CDR1, SEQID NO:362 as CDR2, and SEQ ID NO:363 as CDR3 of SEQ ID NO:285.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesS128; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:286. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:286. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:364 as CDR1, SEQ ID NO:365 as CDR2, and SEQ IDNO:366 as CDR3 of SEQ ID NO:286. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:548 as CDR1,SEQ ID NO:365 as CDR2, and SEQ ID NO:549 as CDR3 of SEQ ID NO:286. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:286 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:286 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:287. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:286 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:287, which includes amino acid sequences SEQ ID NO:367 as CDR1, SEQID NO:368 as CDR2, and SEQ ID NO:369 as CDR3 of SEQ ID NO:287.

In certain embodiments, the present invention provides an antigenbinding site that binds to a unique epitope on human CD33 that includesS128; the antigen binding site includes a heavy chain variable domainincluding an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:288. In some embodiments, the antibody heavy chainvariable domain is at least 95% identical to SEQ ID NO:288. In someembodiments, the heavy chain variable domain incorporates amino acidsequences SEQ ID NO:370 as CDR1, SEQ ID NO:371 as CDR2, and SEQ IDNO:372 as CDR3 of SEQ ID NO:288. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:550 as CDR1,SEQ ID NO:371 as CDR2, and SEQ ID NO:551 as CDR3 of SEQ ID NO:288. Incertain embodiments, the antibody heavy chain variable domain whichincludes an amino acid sequence at least 90% (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:288 is combined with a light chain variable domainto form an antigen-binding site capable of binding to CD33. For example,an antibody heavy chain variable domain at least 90% (e.g., at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to theamino acid sequence of SEQ ID NO:288 can be paired with an antibodylight chain variable domain at least 90% (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acidsequence of SEQ ID NO:289. In certain embodiments, an antibody heavychain variable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:288 can be paired with an antibody light chain variabledomain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:289, which includes amino acid sequences SEQ ID NO:373 as CDR1, SEQID NO:374 as CDR2, and SEQ ID NO:375 as CDR3 of SEQ ID NO:289.

An Antigen Binding Site Binds to the V Domain of Human CD33 in aGlycosylation Sensitive Manner

In one aspect, the present invention provides an antigen binding siteincluding a heavy chain variable domain that binds to the V domain ofhuman CD33 in a glycosylation sensitive manner, e.g., binds to the Vdomain of CD33 only when the V domain is deglycosylated.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope in the V domain of human CD33 onlywhen the V domain is deglycosylated; the antigen binding site includes aheavy chain variable domain including an amino acid sequence at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:278. In someembodiments, the antibody heavy chain variable domain is at least 95%identical to SEQ ID NO:278. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:340 as CDR1,SEQ ID NO:341 as CDR2, and SEQ ID NO:342 as CDR3 of SEQ ID NO:278. Insome embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:540 as CDR1, SEQ ID NO:341 as CDR2, and SEQ IDNO:541 as CDR3 of SEQ ID NO:278. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:278 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:278 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:279. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:278 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:279, which includes amino acidsequences SEQ ID NO:343 as CDR1, SEQ ID NO:344 as CDR2, and SEQ IDNO:345 as CDR3 of SEQ ID NO:279.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope in the V domain of human CD33 onlywhen the V domain is deglycosylated; the antigen binding site includes aheavy chain variable domain including an amino acid sequence at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:282. In someembodiments, the antibody heavy chain variable domain is at least 95%identical to SEQ ID NO:282. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:352 as CDR1,SEQ ID NO:353 as CDR2, and SEQ ID NO:354 as CDR3 of SEQ ID NO:282. Insome embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:544 as CDR1, SEQ ID NO:353 as CDR2, and SEQ IDNO:545 as CDR3 of SEQ ID NO:282. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:282 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:282 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:283. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:282 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:283, which includes amino acidsequences SEQ ID NO:355 as CDR1, SEQ ID NO:356 as CDR2, and SEQ IDNO:357 as CDR3 of SEQ ID NO:283.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope in the V domain of human CD33 onlywhen the V domain is deglycosylated; the antigen binding site includes aheavy chain variable domain including an amino acid sequence at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:284. In someembodiments, the antibody heavy chain variable domain is at least 95%identical to SEQ ID NO:284. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:358 as CDR1,SEQ ID NO:359 as CDR2, and SEQ ID NO:360 as CDR3 of SEQ ID NO:284. Insome embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:546 as CDR1, SEQ ID NO:359 as CDR2, and SEQ IDNO:360 as CDR3 of SEQ ID NO:547. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:284 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:284 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:285. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:284 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:285, which includes amino acidsequences SEQ ID NO:361 as CDR1, SEQ ID NO:362 as CDR2, and SEQ IDNO:363 as CDR3 of SEQ ID NO:285.

In certain embodiments, the present invention provides an antigenbinding site that binds an epitope in the V domain of human CD33 onlywhen the V domain is deglycosylated; the antigen binding site includes aheavy chain variable domain including an amino acid sequence at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:288. In someembodiments, the antibody heavy chain variable domain is at least 95%identical to SEQ ID NO:288. In some embodiments, the heavy chainvariable domain incorporates amino acid sequences SEQ ID NO:370 as CDR1,SEQ ID NO:371 as CDR2, and SEQ ID NO:372 as CDR3 of SEQ ID NO:288. Insome embodiments, the heavy chain variable domain incorporates aminoacid sequences SEQ ID NO:550 as CDR1, SEQ ID NO:371 as CDR2, and SEQ IDNO:551 as CDR3 of SEQ ID NO:288. In certain embodiments, the antibodyheavy chain variable domain which includes an amino acid sequence atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:288 iscombined with a light chain variable domain to form an antigen-bindingsite capable of binding to CD33. For example, an antibody heavy chainvariable domain at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQID NO:288 can be paired with an antibody light chain variable domain atleast 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) identical to the amino acid sequence of SEQ ID NO:289. Incertain embodiments, an antibody heavy chain variable domain at least90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:288 can bepaired with an antibody light chain variable domain at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identicalto the amino acid sequence of SEQ ID NO:289, which includes amino acidsequences SEQ ID NO:373 as CDR1, SEQ ID NO:374 as CDR2, and SEQ IDNO:375 as CDR3 of SEQ ID NO:289.

An Antigen Binding Site that Binds to the Extracellular Domain in HumanCD33 and/or Cyno CD33, Irrespective of the Glycosylation Profile of theTargeted CD33

In one aspect, the present invention provides an antigen binding siteincluding a heavy chain variable domain which includes an amino acidsequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 266, 268, 270, 272, 274, 276, 278, 280,282, 284, 286, 288, 290, 292, 294, 296, 298, 300, or 302, which binds tothe extracellular domain in human CD33 irrespective of the glycosylationprofile of the targeted CD33.

In certain embodiments, the present invention provides an antigenbinding site including a heavy chain variable domain that binds to theextracellular domain in human CD33 and/or cyno CD33, such that theepitopes are unique compared to the epitopes targeted by one or moreknown anti-CD33 antibodies in the art. In certain embodiments, thepresent invention provides an antigen binding site including a heavychain variable domain that binds to the extracellular domain in humanCD33 and/or cyno CD33, which shows human or Cynomolgus/Rhesus (cyno)CD33 cross reactivity and high affinity binding to the target CD33.

A Second Antigen Binding Site Same or Different from the Antigen-BindingSite that Binds Human CD33

In certain embodiments, the present invention provides a protein thatincludes a human CD33 antigen-binding site including a heavy chainvariable domain, which includes an amino acid sequence at least 90%(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, 294, 296, 298, 300, or 302, and further comprises asecond antigen binding site same or different from the antigen-bindingsite that binds to human CD33.

Proteins with Antigen-Binding Sites

An antibody heavy chain variable domain of SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,286, 288, 290, 292, 294, 296, 298, 300, and/or 302 can optionally becoupled to an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to an antibodyconstant region, such as an IgG constant region including hinge, CH2 andCH3 domains with or without a CH1 domain. In some embodiments, the aminoacid sequence of the constant region is at least 90% (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a humanantibody constant region, such as an human IgG1 constant region, an IgG2constant region, IgG3 constant region, or IgG4 constant region. In someother embodiments, the amino acid sequence of the constant region is atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to an antibody constant region from another mammal, suchas rabbit, dog, cat, mouse, or horse. One or more mutations can beincorporated into the constant region as compared to human IgG1 constantregion, for example at Q347, Y349, L351, 5354, E356, E357, K360, Q362,5364, T366, L368, K370, N390, K392, T394, D399, 5400, D401, F405, Y407,K409, T411 and/or K439. Exemplary substitutions include, for example,Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K,L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E,S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W,T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V,K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R,D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D,T411E, K439D, and K439E.

In certain embodiments, mutations that can be incorporated into the CH1of a human IgG1 constant region may be at amino acid V125, F126, P127,T135, T139, A140, F170, P171, and/or V173. In certain embodiments,mutations that can be incorporated into the Cκ of a human IgG1 constantregion may be at amino acid E123, F116, 5176, V163, S174, and/or T164.

II. Multi-Specific Binding Proteins

In certain embodiments, the present invention provides anantigen-binding site in a protein (e.g., a multi-specific bindingprotein) that binds to CD33 on a cancer cell, and the NKG2D receptor andCD16 receptor on natural killer cells to activate the natural killercell. As used herein, the term “antibody” encompasses proteins (e.g.,multi-specific binding proteins) that comprise one or moreantigen-binding sites (e.g., an antigen-binding site that binds CD33),and is not limited to single-specific antibodies. In certainembodiments, the protein (e.g., multi-specific binding protein) orantibody is a trispecific antibody, also called Trispecific NK cellEngagement Therapy (TriNKET). The protein (e.g., a multi-specificbinding protein) is useful in the pharmaceutical compositions andtherapeutic methods described herein. Binding of the protein includingan antigen-binding site that binds to CD33, and to NKG2D receptor andCD16 receptor on natural killer cell enhances the activity of thenatural killer cell toward destruction of a cancer cell. Binding of theprotein including an antigen-binding site that binds to CD33 (e.g., amulti-specific binding protein) on a cancer cell brings the cancer cellinto proximity to the natural killer cell, which facilitates direct andindirect destruction of the cancer cell by the natural killer cell.Further description of exemplary multi-specific binding proteins isprovided below.

In certain embodiments of the present disclosure, the first component ofthe multi-specific binding proteins binds to CD33-expressing cells,which can include but are not limited to AML, myelodysplastic syndromes,chronic myelomonocytic leukemia, myeloid blast crisis of chronic myeloidleukemia, and ALLs.

In certain embodiments of the present disclosure, the second componentof the multi-specific binding proteins binds to NKG2Dreceptor-expressing cells, which can include but are not limited to NKcells, γδ T cells and CD8⁺αβ T cells. Upon NKG2D binding, themulti-specific binding proteins may block natural ligands, such as ULBP6and MICA, from binding to NKG2D and activating NKG2D receptors.

In certain embodiments of the present disclosure, the third componentfor the multi-specific binding proteins binds to cells expressing CD16,an Fc receptor on the surface of leukocytes including natural killercells, macrophages, neutrophils, eosinophils, mast cells, and folliculardendritic cells.

Another aspect of the present invention provides a protein comprising anantigen-binding site that binds NKG2D, the antigen-binding sitecomprising a heavy chain variable domain comprising:

CDR1 comprising the amino acid sequence of SYSMN [SEQ ID NO:192];

CDR2 comprising the amino acid sequence of SISSSSSYIYYADSVKG [SEQ IDNO:112]; and

CDR3 comprising the amino acid sequence of GAPXGAAAGWFDP [SEQ IDNO:527], wherein X is A, V, L, I, P, F, W, G, S, T, C, N, Q, or Y; and alight chain variable domain comprising:

CDR1 comprising the amino acid sequence of RASQGISSWLA [SEQ ID NO:114],

CDR2 comprising the amino acid sequence of AASSLQS [SEQ ID NO:115], and

CDR3 comprising the amino acid sequence of QQGVSFPRT [SEQ ID NO:116].

In certain embodiments, X is A, V, L, I, P, F, or W. In certainembodiments, X is V, L, or I. In certain embodiments, the amino acidsequence of CDR3 in the heavy chain variable domain comprises thesequence of SEQ ID NO:123. In certain embodiments, the amino acidsequence of CDR3 in the heavy chain variable domain comprises thesequence of SEQ ID NO:195, SEQ ID NO:588, SEQ ID NO:591, SEQ ID NO:594,or SEQ ID NO:597.

In certain embodiments, the antigen-binding site comprises a heavy chainvariable domain comprising an amino acid sequence at least 90% (e.g.,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the aminoacid sequence of SEQ ID NO:191; and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence ofSEQ ID NO:81. In certain embodiments, the antigen-binding site comprisesa heavy chain variable domain comprising the amino acid sequence of SEQID NO:191; and a light chain variable domain comprising the amino acidsequence of SEQ ID NO:81.

In certain embodiments, the antigen-binding site that binds NKG2D is inthe form of an Fab fragment. In certain embodiments, the antigen-bindingsite that binds NKG2D is in the form of an scFv.

In certain embodiments, the present invention provides a proteincomprising (a) a first antigen-binding site comprising an Fab fragmentthat binds NKG2D as disclosed herein; (b) a second antigen-binding sitecomprising a single-chain variable fragment (scFv) that binds a tumorassociated antigen (e.g., CD33); and (c) an antibody Fc domain or aportion thereof sufficient to bind CD16, or a third antigen-binding sitethat binds CD16.

The multi-specific binding proteins described herein can take variousformats. For example, one format is a heterodimeric, multi-specificantibody which includes a first immunoglobulin heavy chain, a firstimmunoglobulin light chain, a second immunoglobulin heavy chain and asecond immunoglobulin light chain. The first immunoglobulin heavy chainincludes a first Fc (hinge-CH2-CH3) domain, a first heavy chain variabledomain and optionally a first CH1 heavy chain domain. The firstimmunoglobulin light chain includes a first light chain variable domainand a first light chain constant domain. The first immunoglobulin lightchain, together with the first immunoglobulin heavy chain, forms anantigen-binding site that binds CD33. The second immunoglobulin heavychain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chainvariable domain and optionally a second CH1 heavy chain domain. Thesecond immunoglobulin light chain includes a second light chain variabledomain and a second light chain constant domain. The secondimmunoglobulin light chain, together with the second immunoglobulinheavy chain, forms an antigen-binding site that binds NKG2D. The firstFc domain and second Fc domain together are able to bind to CD16.

Another exemplary format involves a heterodimeric, multi-specificantibody which includes a first immunoglobulin heavy chain, a secondimmunoglobulin heavy chain and an immunoglobulin light chain. The firstimmunoglobulin heavy chain includes a first Fc (hinge-CH2-CH3) domainfused via either a linker or an antibody hinge to a single-chainvariable fragment (scFv) composed of a heavy variable domain and lightchain variable domain which pair and bind CD33 or NKG2D. The secondimmunoglobulin heavy chain includes a second Fc (hinge-CH2-CH3) domain,a second heavy chain variable domain and optionally a CH1 heavy chaindomain. The immunoglobulin light chain includes a light chain variabledomain and a constant light chain domain. The second immunoglobulinheavy chain pairs with the immunoglobulin light chain and binds to NKG2Dor CD33. The first Fc domain and the second Fc domain together are ableto bind to CD16.

One or more additional binding motifs may be fused to the C-terminus ofthe constant region CH3 domain, optionally via a linker sequence. Incertain embodiments, the antigen-binding site could be a single-chain ordisulfide-stabilized variable region (scFv) or could form a tetravalentor trivalent molecule.

In some embodiments, the multi-specific binding protein is in theTriomab form, which is a trifunctional, bispecific antibody thatmaintains an IgG-like shape. This chimera consists of two halfantibodies, each with one light and one heavy chain, that originate fromtwo parental antibodies.

In some embodiments, the multi-specific binding protein is the KiHCommon Light Chain (LC) form, which involves the knobs-into-holes (KIHs)technology. The KIH involves engineering C_(H)3 domains to create eithera “knob” or a “hole” in each heavy chain to promote heterodimerization.The concept behind the “Knobs-into-Holes (KiH)” Fc technology was tointroduce a “knob” in one CH3 domain (CH3A) by substitution of a smallresidue with a bulky one (e.g., T366W_(CH3A) in EU numbering). Toaccommodate the “knob,” a complementary “hole” surface was created onthe other CH3 domain (CH3B) by replacing the closest neighboringresidues to the knob with smaller ones (e.g., T366S/L368A/Y407V_(CH3B)).The “hole” mutation was optimized by structured-guided phage libraryscreening (Atwell S, Ridgway J B, Wells J A, Carter P., Stableheterodimers from remodeling the domain interface of a homodimer using aphage display library, J. Mol. Biol. (1997) 270(1):26-35). X-ray crystalstructures of KiH Fc variants (Elliott J M, Ultsch M, Lee J, Tong R,Takeda K, Spiess C, et al., Antiparallel conformation of knob and holeaglycosylated half-antibody homodimers is mediated by a CH2-CH3hydrophobic interaction. J. Mol. Biol. (2014) 426(9):1947-57; Mimoto F,Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure ofa novel asymmetrically engineered Fc variant with improved affinity forFcgammaRs. Mol. Immunol. (2014) 58(1):132-8) demonstrated thatheterodimerization is thermodynamically favored by hydrophobicinteractions driven by steric complementarity at the inter-CH3 domaincore interface, whereas the knob-knob and the hole-hole interfaces donot favor homodimerization owing to steric hindrance and disruption ofthe favorable interactions, respectively.

In some embodiments, the multi-specific binding protein is in thedual-variable domain immunoglobulin (DVD-Ig™) form, which combines thetarget binding domains of two monoclonal antibodies via flexiblenaturally occurring linkers, and yields a tetravalent IgG-like molecule.

In some embodiments, the multi-specific binding protein is in theOrthogonal Fab interface (Ortho-Fab) form. In the ortho-Fab IgG approach(Lewis S M, Wu X, Pustilnik A, Sereno A, Huang F, Rick H L, et al.,Generation of bispecific IgG antibodies by structure-based design of anorthogonal Fab interface. Nat. Biotechnol. (2014) 32(2):191-8),structure-based regional design introduces complementary mutations atthe LC and HC_(VH-CH1) interface in only one Fab, without any changesbeing made to the other Fab.

In some embodiments, the multi-specific binding protein is in the 2-in-1Ig format. In some embodiments, the multi-specific binding protein is inthe ES form, which is a heterodimeric construct containing two differentFabs binding to targets 1 and target 2 fused to the Fc.Heterodimerization is ensured by electrostatic steering mutations in theFc.

In some embodiments, the multi-specific binding protein is in theκλ-Body form, which is an heterodimeric constructs with two differentFabs fused to Fc stabilized by heterodimerization mutations: Fab 1targeting antigen 1 contains kappa LC, while second Fab targetingantigen 2 contains lambda LC. FIG. 30A is an exemplary representation ofone form of a κλ-Body; FIG. 30B is an exemplary representation ofanother κλ-Body.

In some embodiments, the multi-specific binding protein is in Fab ArmExchange form (antibodies that exchange Fab arms by swapping a heavychain and attached light chain (half-molecule) with a heavy-light chainpair from another molecule, which results in bispecific antibodies). Insome embodiments, the multi-specific binding protein is in the SEED Bodyform. The strand-exchange engineered domain (SEED) platform was designedto generate asymmetric and bispecific antibody-like molecules, acapability that expands therapeutic applications of natural antibodies.This protein engineered platform is based on exchanging structurallyrelated sequences of immunoglobulin within the conserved CH3 domains.The SEED design allows efficient generation of AG/GA heterodimers, whiledisfavoring homodimerization of AG and GA SEED CH3 domains. (Muda M. etal., Protein Eng. Des. Sel. (2011, 24(5):447-54)). In some embodiments,the multi-specific binding protein is in the LuZ-Y form, in which aleucine zipper is used to induce heterodimerization of two differentHCs. (Wranik, B J. et al., J. Biol. Chem. (2012), 287:43331-9).

In some embodiments, the multi-specific binding protein is in theCov-X-Body form. In bispecific CovX-Bodies, two different peptides arejoined together using a branched azetidinone linker and fused to thescaffold antibody under mild conditions in a site-specific manner.Whereas the pharmacophores are responsible for functional activities,the antibody scaffold imparts long half-life and Ig-like distribution.The pharmacophores can be chemically optimized or replaced with otherpharmacophores to generate optimized or unique bispecific antibodies.(Doppalapudi V R et al., PNAS (2010), 107(52); 22611-22616).

In some embodiments, the multi-specific binding protein is in anOasc-Fab heterodimeric form that includes Fab binding to target 1, andscFab binding to target 2 fused to Fc. Heterodimerization is ensured bymutations in the Fc.

In some embodiments, the multi-specific binding protein is in a DuetMabform, which is an heterodimeric construct containing two different Fabsbinding to antigens 1 and 2, and Fc stabilized by heterodimerizationmutations. Fab 1 and 2 contain differential S-S bridges that ensurecorrect LC and HC pairing.

In some embodiments, the multi-specific binding protein is in a CrossmAbform, which is an heterodimeric construct with two different Fabsbinding to targets 1 and 2, fused to Fc stabilized byheterodimerization. CL and CH1 domains and VH and VL domains areswitched, e.g., CH1 is fused in-line with VL, while CL is fused in-linewith VH.

In some embodiments, the multi-specific binding protein is in a Fit-Igform, which is a homodimeric constructs where Fab binding to antigen 2is fused to the N terminus of HC of Fab that binds to antigen 1. Theconstruct contains wild-type Fc.

Additional formats of the multi-specific binding proteins can be devisedby combining various formats of CD33 binding-fragments described herein.

In certain embodiments of the present disclosure, the third componentfor the multi-specific binding proteins is an antibody constant region.In certain embodiments, each of the two immunoglobulin heavy chains ofthe antibody constant region includes a constant region with an aminoacid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to human IgG1 constant region. Incertain embodiments, the amino acid sequence of one polypeptide chain ofthe antibody constant region differs from the amino acid sequence of anIgG1 constant region at one or more positions selected from the groupconsisting of Q347, Y349, L351, 5354, E356, E357, K360, Q362, 5364,T366, L368, K370, K392, T394, D399, 5400, D401, F405, Y407, K409, T411and K439; and the amino acid sequence of the other polypeptide chain ofthe antibody constant region differs from the amino acid sequence of anIgG1 constant region at one or more positions selected from the groupconsisting of Q347, Y349, L351, S354, E356, E357, S364, T366, L368,K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411 and K439.

In certain embodiments of the present disclosure, the NKG2D-antigenbinding site comprises:

(1) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:81 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:82[ADI-29379];

(2) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:83 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:84[ADI-29463];

(3) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:85 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:86[ADI-27744];

(4) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:87 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:88[ADI-27749];

(5) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:191 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:88[A49MI]; or

(6) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:89 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:90[ADI-29378].

In certain embodiments of the present disclosure, the NKG2D-antigenbinding site comprises:

(1) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:124 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:125[ADI-27705];

(2) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:129 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:130[ADI-27724];

(3) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:131 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:132[ADI-27740];

(4) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:133 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:134[ADI-27741];

(5) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:135 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:136[ADI-27743];

(6) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:137 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:138[ADI-28153];

(7) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:139 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ ID NO:140[ADI-28226 (C26)];

(8) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:141 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:142;

(9) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:143 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:144;

(10) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:145 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:146;

(11) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:147 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:148;

(12) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:149 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:150;

(13) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:151 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:152;

(14) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:153 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:154;

(15) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:155 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:156;

(16) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:157 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:158;

(17) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:159 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:160;

(18) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:161 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:162;

(19) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:163 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:164;

(20) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:165 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:166;

(21) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:167 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:168;

(22) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:175 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:176;

(23) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:583 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:584; or

(24) a heavy chain variable domain comprising an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:585 and a light chain variable domaincomprising an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a SEQ IDNO:580.

Table 2 lists peptide sequences of heavy chain variable domains andlight chain variable domains that, in combination, can bind to NKG2D.Unless indicated otherwise, the CDR sequences provided in Table 2 aredetermined under Kabat. The NKG2D-binding domains can vary in theirbinding affinity to NKG2D, nevertheless, they all activate human NKG2Dand NK cells.

TABLE 2 Heavy chain variable region Light chain variable region Clonesamino acid sequence amino acid sequence ADI- QVQLVQSGAEVKKPGASVKVSCKASEIVMTQSPATLSVSPGERATLS 29379 GYTFTSYYMHWVRQAPGQGLEWMGICRASQSVSSNLAWYQQKPGQA (E79) INPSGGSTSYAQKFQGRVTMTRDTSTSPRLLIYGASTRATGIPARFSGSG TVYMELSSLRSEDTAVYYCARGAPNYSGTEFTLTISSLQSEDFAVYYCQ GDTTHDYYYMDVWGKGTTVTVSS QYDDWPFTFGGGTKVEIK[SEQ ID NO: 81] [SEQ ID NO: 82] CDR1: YTFTSYYMH [SEQ ID NO: 93]CDR1: RASQSVSSNLA (non-Kabat) or SYYMH [SEQ ID NO: 566] [SEQ ID NO: 96]CDR2: IINPSGGSTSYAQKFQG CDR2: GASTRAT [SEQ ID NO: 94] [SEQ ID NO: 97]CDR3: ARGAPNYGDTTHDYYYMDV CDR3: QQYDDWPFT [SEQ ID NO: 95] (non-Kabat) or[SEQ ID NO: 98] GAPNYGDTTHDYYYMDV [SEQ ID NO: 567] ADI-QVQLVQSGAEVKKPGASVKVSCKAS EIVLTQSPGTLSLSPGERATLSC 29463GYTFTGYYMHWVRQAPGQGLEWMG RASQSVSSNLAWYQQKPGQAP (F63)WINPNSGGTNYAQKFQGRVTMTRDT RLLIYGASTRATGIPARFSGSGSSISTAYMELSRLRSDDTAVYYCARDT GTEFTLTISSLQSEDFAVYYCQGEYYDTDDHGMDVWGQGTTVTVSS QDDYWPPTFGGGTKVEIK [SEQ ID NO: 83][SEQ ID NO: 84] CDR1: YTFTGYYMH [SEQ ID NO: 99] CDR1: RASQSVSSNLA(non-Kabat) or GYYMH [SEQ ID [SEQ ID NO: 102] NO: 568] CDR2: GASTRATCDR2: WINPNSGGTNYAQKFQG [SEQ ID NO: 103] [SEQ ID NO: 100]CDR3: QQDDYWPPT CDR3: ARDTGEYYDTDDHGMDV [SEQ ID NO: 104][SEQ ID NO: 101] (non-Kabat) or DTGEYYDTDDHGMDV [SEQ ID NO: 569] ADI-EVQLLESGGGLVQPGGSLRLSCAASG DIQMTQSPSSVSASVGDRVTIT 27744FTFSSYAMSWVRQAPGKGLEWVSAIS CRASQGIDSWLAWYQQKPGK (A44)GSGGSTYYADSVKGRFTISRDNSKNT APKLLIYAASSLQSGVPSRFSGSLYLQMNSLRAEDTAVYYCAKDGGYY GSGTDFTLTISSLQPEDFATYYC DSGAGDYWGQGTLVTVSSQQGVSYPRTFGGGTKVEIK [SEQ ID NO: 85] [SEQ ID NO: 86]CDR1: FTFSSYAMS [SEQ ID NO: 105] CDR1: RASQGIDSWLA(non-Kabat) or SYAMS [SEQ ID NO: 570] [SEQ ID NO: 108]CDR2: AISGSGGSTYYADSVKG CDR2: AASSLQS [SEQ ID NO: 106] [SEQ ID NO: 109]CDR3: AKDGGYYDSGAGDY CDR3: QQGVSYPRT [SEQ ID NO: 107] (non-Kabat) or[SEQ ID NO: 110] DGGYYDSGAGDY [SEQ ID NO: 571] ADI-EVQLVESGGGLVKPGGSLRLSCAASG DIQMTQSPSSVSASVGDRVTIT 27749FTFSSYSMNWVRQAPGKGLEWVSSIS CRASQGISSWLAWYQQKPGKA (A49)SSSSYIYYADSVKGRFTISRDNAKNSL PKLLIYAASSLQSGVPSRFSGSGYLQMNSLRAEDTAVYYCARGAPMGA SGTDFTLTISSLQPEDFATYYCQ AAGWFDPWGQGTLVTVSSQGVSFPRTFGGGTKVEIK [SEQ ID NO: 87] [SEQ ID NO: 88]CDR1: FTFSSYSMN [SEQ ID NOT 11] or CDR1: RASQGISSWLASYSMN [SEQ ID NO: 92] [SEQ ID NO: 114] CDR2: SISSSSSYIYYADSVKGCDR2: AASSLQS [SEQ ID NO: 112] [SEQ ID NO: 115] CDR3: (non-Kabat)CDR3: QQGVSFPRT ARGAPMGAAAGWFDP [SEQ ID NO: 116] [SEQ ID NO: 113] orGAPMGAAAGWFDP [SEQ ID NO: 93] A49MI EVQLVESGGGLVKPGGSLRLSCAASGDIQMTQSPSSVSASVGDRVTIT FTFSSYSMNWVRQAPGKGLEWVSSIS CRASQGISSWLAWYQQKPGKASSSSYIYYADSVKGRFTISRDNAKNSL PKLLIYAASSLQSGVPSRFSGSGYLQMNSLRAEDTAVYYCARGAPIGA SGTDFTLTISSLQPEDFATYYCQAAGWFDPWGQGTLVTVSS [SEQ ID QGVSFPRTFGGGTKVEIK NO: 91] [SEQ ID NO: 88]CDR1: (non-Kabat) FTFSSYSMN [SEQ CDR1: RASQGISSWLAID NO: 111 ] or SYSMN [SEQ ID NO: 192] [SEQ ID NO: 114]CDR2: SISSSSSYIYYADSVKG CDR2: AASSLQS [SEQ ID NO: 112] [SEQ ID NO: 115]CDR3: (non-Kabat) CDR3: QQGVSFPRT ARGAPIGAAAGWFDP [SEQ ID[SEQ ID NO: 116] NO: 194] or GAPIGAAAGWFDP [SEQ ID NO: 195] A49MQEVQLVESGGGLVKPGGSLRLSCAASG DIQMTQSPSSVSASVGDRVTITFTFSSYSMNWVRQAPGKGLEWVSSIS CRASQGISSWLAWYQQKPGKASSSSYIYYADSVKGRFTISRDNAKNSL PKLLIYAASSLQSGVPSRFSGSGYLQMNSLRAEDTAVYYCARGAPOGA SGTDFTLTISSLQPEDFATYYCQ AAGWFDPWGQGTLVTVSSQGVSFPRTFGGGTKVEIK (SEQ ID NO: 586) (SEQ ID NO: 88)CDR1 (non-Kabat) (SEQ ID NO: 111)- CDR1 (SEQ ID NO: 114)-FTFSSYSMN or CDR1 (SEQ ID NO: 192)- RASQGISSWLA SYSMNCDR2 (SEQ ID NO: 115)- CDR2 (SEQ ID NO: 112)- AASSLQS SISSSSSYIYYADSVKGCDR3 (SEQ ID NO: 116)- CDR3 (non-Kabat) (SEQ ID NO: 587)- QQGVSFPRTARGAPQGAAAGWFDP or CDR3 (SEQ ID NO: 588)-GAPQGAAAGWFDP A49MLEVQLVESGGGLVKPGGSLRLSCAASG DIQMTQSPSSVSASVGDRVTITFTFSSYSMNWVRQAPGKGLEWVSSIS CRASQGISSWLAWYQQKPGKASSSSYIYYADSVKGRFTISRDNAKNSL PKLLIYAASSLQSGVPSRFSGSGYLQMNSLRAEDTAVYYCARGAPLGA SGTDFTLTISSLQPEDFATYYCQ AAGWFDPWGQGTLVTVSSQGVSFPRTFGGGTKVEIK (SEQ ID NO: 589) (SEQ ID NO: 88)CDR1 (non-Kabat) (SEQ ID NO: 111)- CDR1 (SEQ ID NO: 114)-FTFSSYSMN or CDR1 (SEQ ID NO: 192)- RASQGISSWLA SYSMNCDR2 (SEQ ID NO: 115)- CDR2 (SEQ ID NO: 112)- AASSLQS SISSSSSYIYYADSVKGCDR3 (SEQ ID NO: 116)- CDR3 (non-Kabat) (SEQ ID NO: 590)- QQGVSFPRTARGAPLGAAAGWFDP or CDR3 (SEQ ID NO: 591)-GAPLGAAAGWFDP A49MFEVQLVESGGGLVKPGGSLRLSCAASG DIQMTQSPSSVSASVGDRVTITFTFSSYSMNWVRQAPGKGLEWVSSIS CRASQGISSWLAWYQQKPGKASSSSYIYYADSVKGRFTISRDNAKNSL PKLLIYAASSLQSGVPSRFSGSGYLQMNSLRAEDTAVYYCARGAPFGA SGTDFTLTISSLQPEDFATYYCQ AAGWFDPWGQGTLVTVSSQGVSFPRTFGGGTKVEIK (SEQ ID NO: 592) [SEQ ID NO: 88]CDR1 (non-Kabat) (SEQ ID NO: 111)- CDR1: RASQGISSWLAFTFSSYSMN or CDR1 (SEQ ID NO: 192)- [SEQ ID NO: 114] SYSMN CDR2: AASSLQSCDR2 (SEQ ID NO: 112)- [SEQ ID NO: 115] SISSSSSYIYYADSVKGCDR3: QQGVSFPRT CDR3 (non-Kabat) (SEQ ID NO: 593)- [SEQ ID NO: 116]ARGAPFGAAAGWFDP or CDR3 (SEQ ID NO: 594)-GAPFGAAAGWFDP A49MVEVQLVESGGGLVKPGGSLRLSCAASG DIQMTQSPSSVSASVGDRVTITFTFSSYSMNWVRQAPGKGLEWVSSIS CRASQGISSWLAWYQQKPGKASSSSYIYYADSVKGRFTISRDNAKNSL PKLLIYAASSLQSGVPSRFSGSGYLQMNSLRAEDTAVYYCARGAPVGA SGTDFTLTISSLQPEDFATYYCQ AAGWFDPWGQGTLVTVSSQGVSFPRTFGGGTKVEIK (SEQ ID NO: 595) [SEQ ID NO: 88]CDR1 (non-Kabat) (SEQ ID NO: 111)- CDR1: RASQGISSWLAFTFSSYSMN or CDR1 (SEQ ID NO: 192)- [SEQ ID NO: 114] SYSMN CDR2: AASSLQSCDR2 (SEQ ID NO: 112)- [SEQ ID NO: 115] SISSSSSYIYYADSVKGCDR3: QQGVSFPRT CDR3 (non-Kabat) (SEQ ID NO: 596)- [SEQ ID NO: 116]ARGAPVGAAAGWFDP or CDR3 (SEQ ID NO: 597)-GAPVGAAAGWFDP A49-EVQLVESGGGLVKPGGSLRLSCAASG DIQMTQSPSSVSASVGDRVTIT consensusFTFSSYSMNWVRQAPGKGLEWVSSIS CRASQGISSWLAWYQQKPGKASSSSYIYYADSVKGRFTISRDNAKNSL PKLLIYAASSLQSGVPSRFSGSGYLQMNSLRAEDTAVYYCARGAPXGA SGTDFTLTISSLQPEDFATYYCQAAGWFDPWGQGTLVTVSS, wherein X QGVSFPRTFGGGTKVEIK is M, L, I, V, Q, or F[SEQ ID NO: 88] (SEQ ID NO: 91) CDR1: RASQGISSWLACDR1 (non-Kabat) (SEQ ID NO: 111)- [SEQ ID NO: 114]FTFSSYSMN or CDR1 (SEQ ID NO: 192)- CDR2: AASSLQS SYSMN [SEQ ID NO: 115]CDR2 (SEQ ID NO: 112)- CDR3: QQGVSFPRT SISSSSSYIYYADSVKG[SEQ ID NO: 116] CDR3 (non-Kabat) (SEQ ID NO: 92)-ARGAPXGAAAGWFDP or CDR3 (SEQ ID NO: 123)-GAPXGAAAGWFDP,wherein X is M, L, I, V, Q, or F ADI- QVQLVQSGAEVKKPGASVKVSCKASEIVLTQSPATLSLSPGERATLSC 29378 GYTFTSYYMHWVRQAPGQGLEWMGIRASQSVSSYLAWYQQKPGQAP (E78) INPSGGSTSYAQKFQGRVTMTRDTSTSRLLIYDASNRATGIPARFSGSGS TVYMELSSLRSEDTAVYYCAREGAGFGTDFTLTISSLEPEDFAVYYCQ AYGMDYYYMDVWGKGTTVTVSS QSDNWPFTFGGGTKVEIK[SEQ ID NO: 89] [SEQ ID NO: 90] CDR1: YTFTSYYMH [SEQ ID NO: 117]CDR1: RASQSVSSYLA (non-Kabat) or SYYMH [SEQ ID NO: 572] [SEQ ID NO: 120]CDR2: IINPSGGSTSYAQKFQG CDR2: DASNRAT [SEQ ID NO: 118] [SEQ ID NO: 121]CDR3: AREGAGFAYGMDYYYMDV CDR3: QQSDNWPFT [SEQ ID NO: 119] (non-Kabat) or[SEQ ID NO: 122] EGAGFAYGMDYYYMDV [SEQ ID NO: 573] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 27705GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYNSYPITFGGGTKVEIK [SEQ ID NO: 124] [SEQ ID NO: 125]CDR1: GSFSGYYWS [SEQ ID NO: 126] (non-Kabat) or GYYWS [SEQ ID NO: 574]CDR2: EIDHSGSTNYNPSLKS [SEQ ID NO: 127] CDR3: ARARGPWSFDP [SEQ IDNO: 128] (non-Kabat) or ARGPWSFDP [SEQ ID NO: 575] ADI-QVQLQQWGAGLLKPSETLSLTCAVY EIVLTQSPGTLSLSPGERATLSC 27724GGSFSGYYWSWIRQPPGKGLEWIGEI RASQSVSSSYLAWYQQKPGQADHSGSTNYNPSLKSRVTISVDTSKNQF PRLLIYGASSRATGIPDRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTDFTLTISRLEPEDFAVYYC DPWGQGTLVTVSSQQYGSSPITFGGGTKVEIK [SEQ ID NO: 129] [SEQ ID NO: 130] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 27740GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSIGSWLAWYQQKPGKA (A40)DHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSVT AADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYHSFYTFGGGTKVEIK [SEQ ID NO: 131] [SEQ ID NO: 132] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 27741GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSIGSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQSNSYYTFGGGTKVEIK [SEQ ID NO: 133] [SEQ ID NO: 134] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 27743GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYNSYPTFGGGTKVEIK [SEQ ID NO: 135] [SEQ ID NO: 136] ADI-QVQLQQWGAGLLKPSETLSLTCAVY ELQMTQSPSSLSASVGDRVTIT 28153GGSFSGYYWSWIRQPPGKGLEWIGEI CRTSQSISSYLNWYQQKPGQPPDHSGSTNYNPSLKSRVTISVDTSKNQF KLLIYWASTRESGVPDRFSGSGSLKLSSVTAADTAVYYCARARGPWG SGTDFTLTISSLQPEDSATYYCQ FDPWGQGTLVTVSSQSYDIPYTFGQGTKLEIK [SEQ ID NO: 137] [SEQ ID NO: 138] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 28226GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKA (C26)DHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYGSFPITFGGGTKVEIK [SEQ ID NO: 139] [SEQ ID NO: 140] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 28154GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTDFTLTISSLQPDDFATYYC DPWGQGTLVTVSSQQSKEVPWTFGQGTKVEIK [SEQ ID NO: 141] [SEQ ID NO: 142] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29399GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYNSFPTFGGGTKVEIK [SEQ ID NO: 143] [SEQ ID NO: 144] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29401GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSIGSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYDIYPTFGGGTKVEIK [SEQ ID NO: 145] [SEQ ID NO: 146] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29403GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYDSYPTFGGGTKVEIK [SEQ ID NO: 147] [SEQ ID NO: 148] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29405GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYGSFPTFGGGTKVEIK [SEQ ID NO: 149] [SEQ ID NO: 150] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29407GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYQSFPTFGGGTKVEIK [SEQ ID NO: 151] [SEQ ID NO: 152] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29419GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYSSFSTFGGGTKVEIK [SEQ ID NO: 153] [SEQ ID NO: 154] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29421GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYESYSTFGGGTKVEIK [SEQ ID NO: 155] [SEQ ID NO: 156] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29424GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYDSFITFGGGTKVEIK [SEQ ID NO: 157] [SE QID NO: 158] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29425GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYQSYPTFGGGTKVEIK [SEQ ID NO: 159] [SEQ ID NO: 160] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29426GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSIGSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYHSFPTFGGGTKVEIK [SEQ ID NO: 161] [SEQ ID NO: 162] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29429GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSIGSWLAWYQQKPGKADHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYELYSYTFGGGTKVEIK [SEQ ID NO: 163] [SEQ ID NO: 164] ADI-QVQLQQWGAGLLKPSETLSLTCAVY DIQMTQSPSTLSASVGDRVTIT 29447GGSFSGYYWSWIRQPPGKGLEWIGEI CRASQSISSWLAWYQQKPGKA (F47)DHSGSTNYNPSLKSRVTISVDTSKNQF PKLLIYKASSLESGVPSRFSGSGSLKLSSVTAADTAVYYCARARGPWSF SGTEFTLTISSLQPDDFATYYCQ DPWGQGTLVTVSSQYDTFITFGGGTKVEIK [SEQ ID NO: 165] [SEQ ID NO: 166] ADI-QVQLVQSGAEVKKPGSSVKVSCKAS DIVMTQSPDSLAVSLGERATIN 27727GGTFSSYAISWVRQAPGQGLEWMGGI CKSSQSVLYSSNNKNYLAWYQIPIFGTANYAQKFQGRVTITADESTST QKPGQPPKLLIYWASTRESGVPAYMELSSLRSEDTAVYYCARGDSSIR DRFSGSGSGTDFTLTISSLQAED HAYYYYGMDVWGQGTTVTVSSVAVYYCQQYYSTPITFGGGTK [SEQ ID NO: 167] VEIKCDR1: GTFSSYAIS [SEQ ID NO: 169] [SEQ ID NO: 168](non-Kabat) or SYAIS [SEQ ID NO: 576] CDR1: KSSQSVLYSSNNKNYLACDR2: GIIPIFGTANYAQKFQG [SEQ ID [SEQ ID NO: 172] NO: 170]CDR2: WASTRES [SEQ ID CDR3: ARGDSSIRHAYYYYGMDV NO: 173][SEQ ID NO: 171] (non-Kabat) or CDR3: QQYYSTPIT [SEQ IDGDSSIRHAYYYYGMDV [SEQ ID NO: 174] NO: 577] ADI-QLQLQESGPGLVKPSETLSLTCTVSGG EIVLTQSPATLSLSPGERATLSC 29443SISSSSYYWGWIRQPPGKGLEWIGSIY RASQSVSRYLAWYQQKPGQAP (F43)YSGSTYYNPSLKSRVTISVDTSKNQFS RLLIYDASNRATGIPARFSGSGSLKLSSVTAADTAVYYCARGSDRFHPY GTDFTLTISSLEPEDFAVYYCQ FDYWGQGTLVTVSSQFDTWPPTFGGGTKVEIK [SEQ ID NO: 175] [SEQ ID NO: 176] CDR1: GSISSSSYYWGCDR1: RASQSVSRYLA [SEQ ID NO: 177] (non-Kabat) or [SEQ ID NO: 180]SSSYYWG [SEQ ID NO: 578] CDR2: DASNRAT CDR2: SIYYSGSTYYNPSLKS[SEQ ID NO: 581] [SEQ ID NO: 178] CDR3: QQFDTWPPT CDR3: ARGSDRFHPYFDY[SEQ ID NO: 582] [SEQ ID NO: 179] (non-Kabat) orGSDRFHPYFDY [SEQ ID NO: 579] ADI- QVQLQQWGAGLLKPSETLSLTCAVYDIQMTQSPSTLSASVGDRVTIT 29404 GGSFSGYYWSWIRQPPGKGLEWIGEICRASQSISSWLAWYQQKPGKA (F04) DHSGSTNYNPSLKSRVTISVDTSKNQFPKLLIYKASSLESGVPSRFSGSG SLKLSSVTAADTAVYYCARARGPWSFSGTEFTLTISSLQPDDFATYYCE DPWGQGTLVTVSS QYDSYPTFGGGTKVEIK [SEQ ID NO: 583][SEQ ID NO: 584] ADI- QVQLVQSGAEVKKPGSSVKVSCKAS DIVMTQSPDSLAVSLGERATIN28200 GGTFSSYAISWVRQAPGQGLEWMGGI CESSQSLLNSGNQKNYLTWYQIPIFGTANYAQKFQGRVTITADESTST QKPGQPPKPLIYWASTRESGVPAYMELSSLRSEDTAVYYCARRGRKAS DRFSGSGSGTDFTLTISSLQAEDGSFYYYYGMDVWGQGTTVTVSS VAVYYCQNDYSYPYTFGQGTK [SEQ ID NO: 585] LEIK[SEQ ID NO: 580]

The antibody molecule may have a heavy chain constant region chosenfrom, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4,IgM, IgA1, IgA2, IgD, and IgE; particularly, chosen from, e.g., the(e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, andIgG4. In another embodiment, the antibody molecule has a light chainconstant region chosen from, e.g., the (e.g., human) light chainconstant regions of kappa or lambda. The constant region can be altered,e.g., mutated, to modify the properties of the antibody (e.g., toincrease or decrease one or more of: Fc receptor binding, antibodyglycosylation, the number of cysteine residues, effector cell function,and/or complement function). In one embodiment the antibody has effectorfunction and can fix complement. In other embodiments the antibody doesnot recruit effector cells or fix complement. In another embodiment, theantibody has reduced or no ability to bind an Fc receptor. For example,it is an isotype or subtype, fragment or other mutant, which does notsupport binding to an Fc receptor, e.g., it has a mutagenized or deletedFc receptor binding region.

Within the Fc domain, CD16 binding is mediated by the hinge region andthe CH2 domain. For example, within human IgG1, the interaction withCD16 is primarily focused on amino acid residues Asp 265-Glu 269, Asn297-Thr 299, Ala 327-Ile 332, Leu 234-Ser 239, and carbohydrate residueN-acetyl-D-glucosamine in the CH2 domain (see, Sondermann et al.,Nature, 406 (6793):267-273). Based on the known domains, mutations canbe selected to enhance or reduce the binding affinity to CD16, such asby using phage-displayed libraries or yeast surface-displayed cDNAlibraries, or can be designed based on the known three-dimensionalstructure of the interaction.

In some embodiments, the antibody constant domain comprises a CH2 domainand a CH3 domain of an IgG antibody, for example, a human IgG1 antibody.In some embodiments, mutations are introduced in the antibody constantdomain to enable heterdimerization with another antibody constantdomain. For example, if the antibody constant domain is derived from theconstant domain of a human IgG1, the antibody constant domain cancomprise an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to amino acids 234-332of a human IgG1 antibody, and differs at one or more positions selectedfrom the group consisting of Q347, Y349, L351, 5354, E356, E357, K360,Q362, 5364, T366, L368, K370, N390, K392, T394, D399, 5400, D401, F405,Y407, K409, T411, and K439. All the amino acid positions in an Fc domainor hinge region disclosed herein are numbered according to EU numbering.

The assembly of heterodimeric antibody heavy chains can be accomplishedby expressing two different antibody heavy chain sequences in the samecell, which may lead to the assembly of homodimers of each antibodyheavy chain as well as assembly of heterodimers. Promoting thepreferential assembly of heterodimers can be accomplished byincorporating different mutations in the CH3 domain of each antibodyheavy chain constant region as shown in U.S. Ser. No. 13/494,870, U.S.Ser. No. 16/028,850, U.S. Ser. No. 11/533,709, U.S. Ser. No. 12/875,015,U.S. Ser. No. 13/289,934, U.S. Ser. No. 14/773,418, U.S. Ser. No.12/811,207, U.S. Ser. No. 13/866,756, U.S. Ser. No. 14/647,480, and U.S.Ser. No. 14/830,336. For example, mutations can be made in the CH3domain based on human IgG1 and incorporating distinct pairs of aminoacid substitutions within a first polypeptide and a second polypeptidethat allow these two chains to selectively heterodimerize with eachother. The positions of amino acid substitutions illustrated below areall numbered according to the EU index as in Kabat.

In one scenario, an amino acid substitution in the first polypeptidereplaces the original amino acid with a larger amino acid, selected fromarginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W), and atleast one amino acid substitution in the second polypeptide replaces theoriginal amino acid(s) with a smaller amino acid(s), chosen from alanine(A), serine (S), threonine (T), or valine (V), such that the largeramino acid substitution (a protuberance) fits into the surface of thesmaller amino acid substitutions (a cavity). For example, onepolypeptide can incorporate a T366W substitution, and the other canincorporate three substitutions including T366S, L368A, and Y407V.

An antibody heavy chain variable domain of the invention can optionallybe coupled to an amino acid sequence at least 90% (e.g., 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to an antibodyconstant region, such as an IgG constant region including hinge, CH2 andCH3 domains with or without CH1 domain. In some embodiments, the aminoacid sequence of the constant region is at least 90% (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a humanantibody constant region, such as an human IgG1 constant region, an IgG2constant region, IgG3 constant region, or IgG4 constant region. In someother embodiments, the amino acid sequence of the constant region is atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to an antibody constant region from another mammal, suchas rabbit, dog, cat, mouse, or horse. One or more mutations can beincorporated into the constant region as compared to human IgG1 constantregion, for example at Q347, Y349, L351, 5354, E356, E357, K360, Q362,5364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407,K409, T411 and/or K439. Exemplary substitutions include, for example,Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K,L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E,S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W,T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V,K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R,D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D,T411E, K439D, and K439E.

In certain embodiments, mutations that can be incorporated into the CH1of a human IgG1 constant region may be at amino acid V125, F126, P127,T135, T139, A140, F170, P171, and/or V173. In certain embodiments,mutations that can be incorporated into the Cκ of a human IgG1 constantregion may be at amino acid E123, F116, S176, V163, S174, and/or T164.

Alternatively, amino acid substitutions could be selected from thefollowing sets of substitutions shown in Table 3.

TABLE 3 First Polypeptide Second Polypeptide Set 1 S364E/F405AY349K/T394F Set 2 S364H/D401K Y349T/T411E Set 3 S364H/T394F Y349T/F405ASet 4 S364E/T394F Y349K/F405A Set 5 S364E/T411E Y349K/D401K Set 6S364D/T394F Y349K/F405A Set 7 S364H/F405A Y349T/T394F Set 8 S364K/E357QL368D/K370S Set 9 L368D/K370S S364K Set 10 L368E/K370S S364K Set 11K360E/Q362E D401K Set 12 L368D/K370S S364K/E357L Set 13 K370SS364K/E357Q Set 14 F405L K409R Set 15 K409R F405L

Alternatively, amino acid substitutions could be selected from thefollowing sets of substitutions shown in Table 4.

TABLE 4 First Polypeptide Second Polypeptide Set 1 K409W D399V/F405T Set2 Y349S E357W Set 3 K360E Q347R Set 4 K360E/K409W Q347R/D399V/F405T Set5 Q347E/K360E/K409W Q347R/D399V/F405T Set 6 Y349S/K409WE357W/D399V/F405T

Alternatively, amino acid substitutions could be selected from thefollowing set of substitutions shown in Table 5.

TABLE 5 First Polypeptide Second Polypeptide Set 1 T366K/L351KL351D/L368E Set 2 T366K/L351K L351D/Y349E Set 3 T366K/L351K L351D/Y349DSet 4 T366K/L351K L351D/Y349E/L368E Set 5 T366K/L351K L351D/Y349D/L368ESet 6 E356K/D399K K392D/K409D

Alternatively, at least one amino acid substitution in each polypeptidechain could be selected from Table 6.

TABLE 6 First Polypeptide Second Polypeptide L351Y, D399R, D399K, S400K,T366V, T366I, T366L, T366M, N390D, S400R, Y407A, Y407I, Y407V N390E,K392L, K392M, K392V, K392F K392D, K392E, K409F, K409W, T411D and T411E

Alternatively, at least one amino acid substitutions could be selectedfrom the following set of substitutions in Table 7, where theposition(s) indicated in the First Polypeptide column is replaced by anyknown negatively-charged amino acid, and the position(s) indicated inthe Second Polypeptide Column is replaced by any knownpositively-charged amino acid.

TABLE 7 First Polypeptide Second Polypeptide K392, K370, K409, or K439D399, E356, or E357

Alternatively, at least one amino acid substitutions could be selectedfrom the following set of in Table 8, where the position(s) indicated inthe First Polypeptide column is replaced by any known positively-chargedamino acid, and the position(s) indicated in the Second PolypeptideColumn is replaced by any known negatively-charged amino acid.

TABLE 8 First Polypeptide Second Polypeptide D399, E356, or E357 K409,K439, K370, or K392

Alternatively, amino acid substitutions could be selected from thefollowing set of in Table 9.

TABLE 9 First Polypeptide Second Polypeptide T350V, L351Y, F405A, andY407V T350V, T366L, K392L, and T394W

Alternatively, or in addition, the structural stability of heterodimericheavy chains within the multi-specific binding proteins can be increasedby introducing S354C on either of the first or second polypeptide chain,and Y349C on the opposing polypeptide chain, which forms an artificialdisulfide bridge within the interface of the two polypeptides.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region atposition T366, and wherein the amino acid sequence of the otherpolypeptide chain of the antibody constant region differs from the aminoacid sequence of an IgG1 constant region at one or more positionsselected from the group consisting of T366, L368 and Y407.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of T366, L368 andY407, and wherein the amino acid sequence of the other polypeptide chainof the antibody constant region differs from the amino acid sequence ofan IgG1 constant region at position T366.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of E357, K360,Q362, 5364, L368, K370, T394, D401, F405, and T411 and wherein the aminoacid sequence of the other polypeptide chain of the antibody constantregion differs from the amino acid sequence of an IgG1 constant regionat one or more positions selected from the group consisting of Y349,E357, S364, L368, K370, T394, D401, F405 and T411.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of Y349, E357,S364, L368, K370, T394, D401, F405 and T411 and wherein the amino acidsequence of the other polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of E357, K360,Q362, S364, L368, K370, T394, D401, F405, and T411.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of L351, D399, 5400and Y407 and wherein the amino acid sequence of the other polypeptidechain of the antibody constant region differs from the amino acidsequence of an IgG1 constant region at one or more positions selectedfrom the group consisting of T366, N390, K392, K409 and T411.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of T366, N390,K392, K409 and T411 and wherein the amino acid sequence of the otherpolypeptide chain of the antibody constant region differs from the aminoacid sequence of an IgG1 constant region at one or more positionsselected from the group consisting of L351, D399, S400 and Y407.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of Q347, Y349,K360, and K409, and wherein the amino acid sequence of the otherpolypeptide chain of the antibody constant region differs from the aminoacid sequence of an IgG1 constant region at one or more positionsselected from the group consisting of Q347, E357, D399 and F405.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of Q347, E357, D399and F405, and wherein the amino acid sequence of the other polypeptidechain of the antibody constant region differs from the amino acidsequence of an IgG1 constant region at one or more positions selectedfrom the group consisting of Y349, K360, Q347 and K409.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of K370, K392, K409and K439, and wherein the amino acid sequence of the other polypeptidechain of the antibody constant region differs from the amino acidsequence of an IgG1 constant region at one or more positions selectedfrom the group consisting of D356, E357 and D399.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of D356, E357 andD399, and wherein the amino acid sequence of the other polypeptide chainof the antibody constant region differs from the amino acid sequence ofan IgG1 constant region at one or more positions selected from the groupconsisting of K370, K392, K409 and K439.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of L351, E356, T366and D399, and wherein the amino acid sequence of the other polypeptidechain of the antibody constant region differs from the amino acidsequence of an IgG1 constant region at one or more positions selectedfrom the group consisting of Y349, L351, L368, K392 and K409.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region at oneor more positions selected from the group consisting of Y349, L351,L368, K392 and K409, and wherein the amino acid sequence of the otherpolypeptide chain of the antibody constant region differs from the aminoacid sequence of an IgG1 constant region at one or more positionsselected from the group consisting of L351, E356, T366 and D399.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region by anS354C substitution and wherein the amino acid sequence of the otherpolypeptide chain of the antibody constant region differs from the aminoacid sequence of an IgG1 constant region by a Y349C substitution.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region by aY349C substitution and wherein the amino acid sequence of the otherpolypeptide chain of the antibody constant region differs from the aminoacid sequence of an IgG1 constant region by an S354C substitution.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region by K360Eand K409W substitutions and wherein the amino acid sequence of the otherpolypeptide chain of the antibody constant region differs from the aminoacid sequence of an IgG1 constant region by Q347R, D399V and F405Tsubstitutions.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region byO347R, D399V and F405T substitutions and wherein the amino acid sequenceof the other polypeptide chain of the antibody constant region differsfrom the amino acid sequence of an IgG1 constant region by K360E andK409W substitutions.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region by aT366W substitutions and wherein the amino acid sequence of the otherpolypeptide chain of the antibody constant region differs from the aminoacid sequence of an IgG1 constant region by T366S, T368A, and Y407Vsubstitutions.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region byT366S, T368A, and Y407V substitutions and wherein the amino acidsequence of the other polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region by aT366W substitution.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region byT350V, L351Y, F405A, and Y407V substitutions and wherein the amino acidsequence of the other polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region byT350V, T366L, K392L, and T394W substitutions.

In certain embodiments of the present disclosure, the amino acidsequence of one polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region byT350V, T366L, K392L, and T394W substitutions and wherein the amino acidsequence of the other polypeptide chain of the antibody constant regiondiffers from the amino acid sequence of an IgG1 constant region byT350V, L351Y, F405A, and Y407V substitutions.

Listed below are examples of CD33-binding F3′-TriNKETs comprising: aCD33-binding single-chain variable fragment (scFv) linked to an Fcdomain via a hinge comprising Ala-Ser; and an NKG2D-binding Fab fragment(“A49,” “A49MI, “A49MQ,” “A49ML,” “A49MF,” or “A49MV”) comprising aheavy chain portion comprising an heavy chain variable domain (SEQ IDNO:87 or SEQ ID NO:191) and a CH1 domain, and a light chain portioncomprising a light chain variable domain (SEQ ID NO:88) and a lightchain constant domain, wherein the heavy chain variable domain isconnected to the CH1 domain, and the CH1 domain is connected to an Fcdomain. The CDR sequences are underlined.

In each of the examples, the Fc domain linked to the CD33-binding scFvcomprises Q347R, D399V, and F405T substitutions for forming aheterodimer with the Fc domain linked to the Fab comprising K360E andK409W substitutions. These substitutions in the Fc domains arebold-underlined in the sequences described below. Alternatively, in anexemplary embodiment, the Fc domain linked to the NKG2D-binding Fabfragment includes the mutations of Q347R, D399V, and F405T, and the Fcdomain linked to the CD33-binding scFv comprises matching mutationsK360E and K409W for forming a heterodimer.

Additionally, the Fc domain linked to the CD33-binding scFv comprisesS354C substitution, and the Fc domain linked to the Fab comprises Y349Csubstitution, thereby stabilizing the interaction between the two Fcdomains via a S-S bridge. These substitutions in the Fc domains arebold-italics-underlined in the sequences below.

Alternatively, in an exemplary embodiment, the Fc domain linked to theNKG2D-binding Fab fragment includes a S354C substitution in the CH3domain, which forms a disulfide bond with a Y349C substitution on the Fclinked to the CD33-binding scFv.

A CD33-binding scFv of the present disclosure can include a heavy chainvariable domain connected to a light chain variable domain with a (G45)₄linker. The scFv is linked to an Fc domain via a hinge comprisingAla-Ser (bolded-underlined). SEQ ID NOs:188, 198, and 206-223 areexemplary sequences of such CD33-binding scFv polypeptides. The V_(L)and V_(H) comprised within the scFv (e.g., SEQ ID NOs:188, 198 or206-223) contain 100V_(L)-44V_(H) S-S bridge (resulting from G100C andG44C substitutions, respectively) (cysteine residues are inbold-italics-underlined in the sequences below). (G45)₄ is thebold-underlined sequence GGGGSGGGGSGGGGSGGGGS [SEQ ID NO:186] in SEQ IDNOs:188, 198, and 206-243.

Exemplary sequences of CD33-binding scFvs linked to an Fc domain via ahinge comprising Ala-Ser are provided below.

Abi scFv (LC-HC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 224] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESFPTFG

G TKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGK

LEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTV TVSS ASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAbi scFv (HC-LC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 225] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGK

LEWVAN IKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS GGGGSGGGGSGGGGSGGGGS DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESFPTFG

GTK VEIK AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAb2 scFv (LC-HC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 226] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLESYPLTFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVL V SDGSF TLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGAb2 scFv (HC-LC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 227] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LEWVAN IKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS GGGGSGGGGSGGGGSGGGGS DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQLESYPLTFG

GTKVEIK AS DKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVL V SDGSF TLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGH76scFv (LC-HC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 197] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFG

G TKVEIK GGGGSGGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMSWVRQAPGK

LEWVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTV TVSS ASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH76 scFv (HC-LC) -Fc (Q347R, D399V, F405T, and S354 Csubstitutions)[SEQ ID NO: 228] EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMSWVRQAPGK

LEWVSA IVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS GGGGSGGGGSGGGGSGGGGS DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFG

GTK VEIK AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG H76 scFc (LC-HC)-Fc (K360E, K409W,and Y349C substitutions) [SEQ ID NO: 243]DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFG

G TKVEIK GGGGSGGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMSWVRQAPGK

LEWVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTV TVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

TLPPSRDELT E NQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS W LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAb4 scFv (LC-HC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 229] DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVALP ITFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQ

LEWMGMINPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWGRGTL VTVSS ASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAb4 scFv (HC-LC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 230] QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYYMHWVRQAPGQ

LEWMGM INPSWGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREAADGFVGERYFDLWGRGTLVTVSS GGGGSGGGGSGGGGSGGGGS DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQDVALPITFG

GT KVEIK AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG107 scFv (LC-HC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO:  187] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGK

LEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVL V SDGSF TLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPG107 scFv (HC-LC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 231] EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGK

LEWVAT IKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS GGGGSGGGGSGGGGSGGGGS DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFG

GTKVEIK AS DK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVL V SDGSF TLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG107scFc (LC-HC)-Fc (K360E, K409W, and Y349C substitutions)[SEQ ID NO: 242] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGK

LEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS AS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

TLPPSRDELT E NQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS WLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGAb6 scFv (LC-HC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 232] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGK

LEWVATIKRDGSEKGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVL V SDGSF TLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGAb6 scFv (HC-LC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 233] EVQLVESGGGLVQPGGSLRLSCAASGFTFPSYWMSWVRQAPGK

LEWVAT IKRDGSEKGYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS GGGGSGGGGSGGGGSGGGGS DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFG

GTKVEIK AS DK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVL V SDGSF TLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGAb7scFv (LC-HC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 234] DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQ

LEWMGIINPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWGKGTTVTVSS AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Ab7scFv (HC-LC)-Fc (Q347R, D399V, F405T,and S354C substitutions) [SEQ ID NO: 235]QVQLVQSGAEVKKPGASVKVSCKASGYTFGTYYMHWVRQAPGQ

LEWMGI INPSRGSTVYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAGYDDEDMDVWGKGTTVTVSS GGGGSGGGGSGGGGSGGGGS DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHSYPLTFG

GTKVEIK AS D KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Ab8 scFv (LC-HC)-Fc (Q347R, D399V, F405T,and S354C substitutions) [SEQ ID NO: 236]DIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFG

G TKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGK

LEWVSSISSSSEGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTV TVSS ASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAb8 scFv (HC-LC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 237] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGK

LEWVSS ISSSSEGIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTVTVSS GGGGSGGGGSGGGGSGGGGS DIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFG

GTK VEIK AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAb9 scFv (LC-HC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 238] DIQMTQSPSSLSASVGDRVTITCRASQVIYSYLNWYQQKPGKAPKLLIYAASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPLTFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LEWVANINTDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDYWGQGTLVTVSS ASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Ab9 scFv (HC-LC)-Fc (Q347R, D399V, F405T,and S354C substitutions) [SEQ ID NO: 239]EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGK

LEWVAN INTDGSEVYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDVGPGIAYQGHFDYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQVIYSYLNWYQQKPGKAPKLLIYAASSLKSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYDTPLTFG

GTKVEIK AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Ab10 scFv (LC-HC)-Fc (Q347R, D399V, F405T,and S354C substitutions) [SEQ ID NO: 240]EIVLTQSPATLSLSPGERATLSCRASHSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPTFG

G TKVEIK GGGGSGGGGSGGGGSGGGGS QLQLQESGPGLVKPSETLSLTCTVSGGSISSTDYYWGWIRQPPGK

LEWIGSIGYSGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQGTTVTVSS AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVL V SDGSF TLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGAb10 scFv (HC-LC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 241] QLQLQESGPGLVKPSETLSLTCTVSGGSISSTDYYWGWIRQPPGK

LEWI GSIGYSGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARETAHDVHGMDVWGQGTTVTVSS GGGGSGGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASHSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYDNLPTFG

GTKVEIK AS DK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVL V SDGSF TLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG

A TriNKET of the present disclosure is A49-F3′-TriNKET-I07, comprising afirst polypeptide chain, named “I07 scFv-Fc,” which comprises aCD33-binding scFv linked to an Fc domain via an Ala-Ser linker; a secondpolypeptide chain, named “A49 VH-CH1-Fc,” which comprises anNKG2D-targeting heavy chain; and a third polypeptide chain, named “A49VL-CL,” which comprises an NKG2D-targeting light chain. The amino acidsequence of I07 scFv-Fc comprises:

107 scFv (LC-HC)-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 187] DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGK

LEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS AS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVL V SDGSF TLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG

The amino acid sequence of the scFv portion of I07 scFv-Fc comprises:

107 scFv [SEQ ID NO: 188]DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFG

GTKVEIK GGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGK

LEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS

A49 VH-CH1-Fc comprises A49 VH [SEQ ID NO:87] linked to a CH1 domain andan Fc domain (including hinge, CH2, and CH3 domains). The amino acidsequence of A49 VH-CH1-Fc comprises:

A49 VH-CH1-Fc (K360E, K409W, and Y349C substitutions) [SEQ ID NO: 189]EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQV

TLPPSRDELT E NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYS WLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GA49 VH-CH1-Fc (Q347R, D399V, F405T, and) S354C substitutions[SEQ ID NO: 244] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VL V SDGSF TLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

A49 VL-CL comprises A49 VL [SEQ ID NO:88] linked to a light chainconstant domain (CL). The amino acid sequence of A49 VL-CL comprises:

A49 VL-CL [SEQ ID NO: 190]DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Another TriNKET of the present disclosure is A49MI-F3′-TriNKET-I07,comprising a first polypeptide chain, named “I07 scFv-Fc,” as describedabove [SEQ ID NO:187]; a second polypeptide chain, named “A49MIVH-CH1-Fc,” that comprises an NKG2D-targeting heavy chain with an Fcdomain; and a third polypeptide chain, named “A49 VL-CL,” as describedabove [SEQ ID NO:190]. A49MI-F3′-TriNKET-I07 is identical toA49-F3′-TriNKET-I07 except for a substitution of I for M in CDR3 of theNKG2D-targeting VH. This substitution is bold-italic (and underlinedbecause it is part of a CDR) in the sequence below. A49MI VH-CH1-Fccomprises A49MI VH [SEQ ID NO:191] linked to a CH1 domain and an Fcdomain (including hinge, CH2, and CH3 domains). The amino acid sequenceof A49MI VH-CH1-Fc comprises:

A49MI VH-CH1-Fc (K360E, K409W, and Y349C substitutions) [SEQ ID NO: 196]EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGA P

GAAAGWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQV

TLPPSRDELT E NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYS WLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GA49MI VH-CH1-Fc (Q347R, D399V, F405T, and S354C substitutions)[SEQ ID NO: 245] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGA P

GAAAGWFDPWGQGTLVTVSSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VL V SDGSF TLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

Another TriNKET of the present disclosure is A49-F3′-TriNKET-I07(si),comprising a first polypeptide chain, named “I07 scFv-Fc(si),” whichcomprises a CD33-binding scFv linked to a silent Fc domain via anAla-Ser linker; a second polypeptide chain, named “A49 VH-CH1-Fc(si),”which comprises an NKG2D-targeting heavy chain with a silent Fc domain;and a third polypeptide chain, named “A49 VL-CL,” as described above[SEQ ID NO: 190]. A49-F3′-TriNKET-I07(si) is identical toA49-F3′-TriNKET-I07 except for L234A, L235A, and P329A substitutions inboth Fc domains. These substitutions are bold-italic in the twosequences below. The amino acid sequence of I07 scFv-Fc(si) comprises:

107 scFv-Fc(si) [SEQ ID NO: 204]DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFG CGTKVEIKGGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKCLEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSS AS DK THTCPPCPAPE

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVL V SDGSF TLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPG

The amino acid sequence of the scFv portion of I07 scFv-Fc(si) isidentical to that of the scFv portion of I07 scFv-Fc as described above[SEQ ID NO:188].

The amino acid sequence of A49 VH-CH1-Fc(si) comprises:

A49 VH-CH1-Fc(si) [SEQ ID NO: 205]EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE

GGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

APIEKTISKAKGQPREP QV

TLPPSRDELT E NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSWLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Another TriNKET of the present disclosure is A49-F3′-TriNKET-H76,comprising a first polypeptide chain, named “H76 scFv-Fc,” thatcomprises a CD33-binding scFv linked to an Fc domain via an Ala-Serlinker; a second polypeptide chain, named “A49 VH-CH1-Fc,” as describedabove [SEQ ID NO:189]; and a third polypeptide chain, named “A49 VL-CL,”as described above [SEQ ID NO:190]. The amino acid sequence of H76scFv-Fc comprises:

H76 scFv-Fc [SEQ ID NO: 197]DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFG

G TKVEIK GGGGSGGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMSWVRQAPGK

LEWVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTV TVSS ASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP R VYTLPP

RDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL V SDGSF T LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

The amino acid sequence of the scFv portion of H76 scFv-Fc comprises:

H76 scFv [SEQ ID NO: 198]DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFG

G TKVEIK GGGGSGGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTFSKYTMSWVRQAPGK

LEWVSAIVGSGESTYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGGPYYDSSGYFVYYGMDVWGQGTTV TVSS

In other embodiments of the present disclosure, the CD33-binding scFv ofthe multi-specific binding protein (e.g., TriNKET) comprises the heavychain variable domain CDR1, CDR2, and CDR3, and light chain variabledomain CDR1, CDR2, and CDR3, of any one of the antibodies provided inTable 1. In certain embodiments, the amino acid sequence of the heavychain variable domain is identical to the VH sequence of an antibody inTable 1 except for a substitution of Cys at position 44, and the aminoacid sequence of the light chain variable domain is identical to the VLsequence of the same antibody except for a substitution of Cys atposition 100. In certain embodiments, the heavy chain variable domain isconnected to the light chain variable domain with a (G4S)₄ linker. Theheavy chain variable domain can be N-terminal to the light chainvariable domain or C-terminal to the light chain variable domain. ThescFv is linked to an Fc domain via a hinge comprising Ala-Ser.

The multi-specific binding proteins described above can be made usingrecombinant DNA technology well known to a skilled person in the art.For example, a first nucleic acid sequence encoding the firstimmunoglobulin heavy chain can be cloned into a first expression vector;a second nucleic acid sequence encoding the second immunoglobulin heavychain can be cloned into a second expression vector; a third nucleicacid sequence encoding the first immunoglobulin light chain can becloned into a third expression vector; a fourth nucleic acid sequenceencoding the second immunoglobulin light chain can be cloned into afourth expression vector; the first, second, third and fourth expressionvectors can be stably transfected together into host cells to producethe multimeric proteins.

To achieve the highest yield of the multi-specific binding proteins,different ratios of the first, second, third and fourth expressionvectors can be explored to determine the optimal ratio for transfectioninto the host cells. After transfection, single clones can be isolatedfor cell bank generation using methods known in the art, such as limiteddilution, ELISA, FACS, microscopy, or Clonepix.

Clones can be cultured under conditions suitable for bio-reactorscale-up and maintained expression of the multi-specific protein. Themulti-specific binding proteins can be isolated and purified usingmethods known in the art including centrifugation, depth filtration,cell lysis, homogenization, freeze-thawing, affinity purification, gelfiltration, ion exchange chromatography, hydrophobic interactionexchange chromatography, and mixed-mode chromatography.

In certain embodiments, the antibody binds CD33 with a K_(D) of 20 nM,15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM orlower, as measured using standard binding assays, for example, surfaceplasmon resonance or bio-layer interferometry. In certain embodimentsthe antibody binds EBI3 from a body fluid, tissue and/or cell of asubject.

Competition assays for determining whether an antibody binds to the sameepitope as, or competes for binding with a disclosed antibody, e.g., theAb1 antibody, the Ab2 antibody, the Ab3 antibody, the Ab4 antibody, orthe Ab5 antibody, are known in the art. Exemplary competition assaysinclude immunoassays (e.g., ELISA assays, RIA assays), surface plasmonresonance (e.g., BIAcore analysis), bio-layer interferometry, and flowcytometry.

Typically, a competition assay involves the use of an antigen (e.g., ahuman CD33 protein or fragment thereof) bound to a solid surface orexpressed on a cell surface, a test CD33-binding antibody and areference antibody. The reference antibody is labeled and the testantibody is unlabeled. Competitive inhibition is measured by determiningthe amount of labeled reference antibody bound to the solid surface orcells in the presence of the test antibody. Usually the test antibody ispresent in excess (e.g., 1×, 5×, 10×, 20× or 100×). Antibodiesidentified by competition assay (e.g., competing antibodies) includeantibodies binding to the same epitope, or similar (e.g., overlapping)epitopes, as the reference antibody, and antibodies binding to anadjacent epitope sufficiently proximal to the epitope bound by thereference antibody for steric hindrance to occur.

A competition assay can be conducted in both directions to ensure thatthe presence of the label does not interfere or otherwise inhibitbinding. For example, in the first direction the reference antibody islabeled and the test antibody is unlabeled, and in the second direction,the test antibody is labeled and the reference antibody is unlabeled.

A test antibody competes with the reference antibody for specificbinding to the antigen if an excess of one antibody (e.g., 1×, 5×, 10×,20× or 100×) inhibits binding of the other antibody, e.g., by at least50%, 75%, 90%, 95% or 99% as measured in a competitive binding assay.

Two antibodies may be determined to bind to the same epitope ifessentially all amino acid mutations in the antigen that reduce oreliminate binding of one antibody reduce or eliminate binding of theother. Two antibodies may be determined to bind to overlapping epitopesif only a subset of the amino acid mutations that reduce or eliminatebinding of one antibody reduce or eliminate binding of the other.

The antibodies disclosed herein may be further optimized (e.g.,affinity-matured) to improve biochemical characteristics includingaffinity and/or specificity, improve biophysical properties includingaggregation, stability, precipitation and/or non-specific interactions,and/or to reduce immunogenicity. Affinity-maturation procedures arewithin ordinary skill in the art. For example, diversity can beintroduced into an immunoglobulin heavy chain and/or an immunoglobulinlight chain by DNA shuffling, chain shuffling, CDR shuffling, randommutagenesis and/or site-specific mutagenesis.

In certain embodiments, isolated human antibodies contain one or moresomatic mutations. In these cases, antibodies can be modified to a humangermline sequence to optimize the antibody (e.g., by a process referredto as germlining).

Generally, an optimized antibody has at least the same, or substantiallythe same, affinity for the antigen as the non-optimized (or parental)antibody from which it was derived. Preferably, an optimized antibodyhas a higher affinity for the antigen when compared to the parentalantibody.

If the antibody is for use as a therapeutic, it can be conjugated to aneffector agent such as a small molecule toxin or a radionuclide usingstandard in vitro conjugation chemistries. If the effector agent is apolypeptide, the antibody can be chemically conjugated to the effectoror joined to the effector as a fusion protein. Construction of fusionproteins is within ordinary skill in the art.

The antibody can be conjugated to an effector moiety such as a smallmolecule toxin or a radionuclide using standard in vitro conjugationchemistries. If the effector moiety is a polypeptide, the antibody canbe chemically conjugated to the effector or joined to the effector as afusion protein. Construction of fusion proteins is within ordinary skillin the art.

In certain embodiments, the protein (e.g., multi-specific bindingprotein) of the present disclosure is not substantially internalized bya CD33-expressing cell. A low level of internalization may improve thepharmacokinetics of the protein, thereby reducing the dose required toengage CD33-expressing target cells with effector cells (e.g., NKcells). Internalization can be measured by any method known in the art,e.g., the methods described in Examples 5 and 10 of the presentdisclosure. For example, in certain embodiments, internalization of theprotein (e.g., multi-specific binding protein) by EOL-1 cells is lowerthan 25%, 30%, 35%, 40%, 45%, or 50% after a two-hour incubation, asassessed by the methods disclosed herein. In certain embodiments,internalization of the protein (e.g., multi-specific binding protein) byEOL-1 cells is lower than 25%, 30%, 35%, 40%, 45%, or 50% after a24-hour incubation, as assessed by the methods disclosed herein. Incertain embodiments, internalization of the protein (e.g.,multi-specific binding protein) by Molm-13 cells is lower than 25%, 30%,35%, 40%, 45%, or 50% after a two-hour incubation, as assessed by themethods disclosed herein.

KHYG-1 cells express surface NKG2D, but do not express CD16. In certainembodiments, TriNKET mediated killings of Molm-13 and THP-1 cells aredependent upon NKG2D-mediated activation of the KHYG-1 effector cells.In certain embodiments, TriNKETs of the present disclosure mediateKHYG-1 effector cell killing of Molm-13 (FIG. 15A), EOL-1 (FIG. 16 ),and THP-1 (FIG. 17A) human AML target cell lines.

In certain embodiments, TriNKETs of the present disclosure mediatecytotoxicity of rested human NK cells against Molm-13 or THP-1 human AMLcells. FIGS. 15B and 38A show that TriNKETs of the present disclosuremediate rested human NK cell killing of Molm-13 human AML cells. Therested human NK effector cell (E) to target cancer cell (T) ratio (E:T)was 10:1 in FIG. 15B and 5:1 in FIG. 38A. The E:T ratio may reflectdifferences in the maximal % lysis.

In certain embodiments, TriNKETs of the present disclosure mediaterested human NK cell killing of EOL-1 human AML cells. FIG. 38B showsthat TriNKETs mediate rested human NK cell killing of EOL-1 cells at anE:T of 5:1. In certain embodiments, TriNKETs of the present disclosuremediate rested human NK cell killing of THP-1 target cells, whichexpress the high-affinity FcγRI. FIGS. 17B, 38C, and 38D show thatTriNKETs mediate rested human NK cell killing of THP-1 human AML cellsusing an E:T of 5:1.

In certain embodiments, TriNKETs of the present disclosure mediate humanCD8⁺ T cell killing of Molm-13 target cells. FIGS. 40A-40B show thatTriNKETs mediate human CD8⁺ T cell killing of Molm-13 cells at an E:T of50:1.

A Protein Comprising an Antigen-Binding Site that Competes with theCD33-Binding Sites Described Herein

In one aspect, the present invention provides a protein that includes anantigen-binding site that competes with the CD33-binding sites describedherein to bind to CD33. In certain embodiments, the present inventionprovides a protein that includes an antigen-binding site that competesfor binding to human and cynomolgus CD33 with an antibody that includesan antibody heavy chain having the amino acid sequence of SEQ ID NO:1and an antibody light chain having the amino acid sequence of SEQ IDNO:2.

In certain embodiments, the present invention provides a protein thatincludes an antigen-binding site that competes for binding to human andcynomolgus CD33 with an antibody that includes an antibody heavy chainhaving the amino acid sequence of SEQ ID NO:3 and an antibody lightchain having the amino acid sequence of SEQ ID NO:4.

In certain embodiments, the present invention provides a protein thatincludes an antigen-binding site that competes for binding to human andcynomolgus CD33 with an antibody that includes an antibody heavy chainhaving the amino acid sequence of SEQ ID NO:5 and an antibody lightchain having the amino acid sequence of SEQ ID NO:6.

In certain embodiments, the present invention provides a protein thatincludes an antigen-binding site that competes for binding to human andcynomolgus CD33 with an antibody that includes an antibody heavy chainhaving the amino acid sequence of SEQ ID NO:7 and an antibody lightchain having the amino acid sequence of SEQ ID NO:8.

In certain embodiments, the present invention provides a protein thatincludes an antigen-binding site that competes for binding to human andcynomolgus CD33 with an antibody that includes an antibody heavy chainhaving the amino acid sequence of SEQ ID NO:9 and an antibody lightchain having the amino acid sequence of SEQ ID NO:10.

In certain embodiments, the present invention provides a protein thatincludes an antigen-binding site that competes for binding to human andcynomolgus CD33 with an antibody that includes an antibody heavy chainhaving the amino acid sequence of SEQ ID NO:11 and an antibody lightchain having the amino acid sequence of SEQ ID NO:12.

In certain embodiments, the present invention provides a protein thatincludes an antigen-binding site that competes for binding to human andcynomolgus CD33 with an antibody that includes an antibody heavy chainhaving the amino acid sequence of SEQ ID NO:13 and an antibody lightchain having the amino acid sequence of SEQ ID NO:14.

In certain embodiments, the present invention provides a protein thatincludes an antigen-binding site that competes for binding to human andcynomolgus CD33 with an antibody that includes an antibody heavy chainhaving the amino acid sequence of SEQ ID NO:15 and an antibody lightchain having the amino acid sequence of SEQ ID NO:16.

In certain embodiments, the present invention provides a protein thatincludes an antigen-binding site that competes for binding to human andcynomolgus CD33 with an antibody that includes an antibody heavy chainhaving the amino acid sequence of SEQ ID NO:17 and an antibody lightchain having the amino acid sequence of SEQ ID NO:18.

In certain embodiments, the present invention provides a protein thatincludes an antigen-binding site that competes for binding to human andcynomolgus CD33 with an antibody that includes an antibody heavy chainhaving the amino acid sequence of SEQ ID NO:19 and an antibody lightchain having the amino acid sequence of SEQ ID NO:20.

In some embodiments, an antigen-binding site of the protein thatcompetes with the CD33-binding sites includes a heavy chain variabledomain having an amino acid sequence at least 50% (e.g., 50%, 60%, 70%,80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:1 and a light chainvariable domain having an amino acid sequence at least at least 50%(e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence of SEQ ID NO:2. Insome embodiments, an antigen-binding site of the protein that competeswith the CD33-binding sites includes a heavy chain variable domainhaving an amino acid sequence at least at least 50% (e.g., 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:3 and a light chainvariable domain having an amino acid sequence at least at least 50%(e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence of SEQ ID NO:4.

In some embodiments, an antigen-binding site of the protein thatcompetes with the CD33-binding sites includes a heavy chain variabledomain having an amino acid sequence at least at least 50% (e.g., 50%,60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:5 and a lightchain variable domain having an amino acid sequence at least at least50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:6.

In some embodiments, an antigen-binding site of the protein thatcompetes with the CD33-binding sites includes a heavy chain variabledomain having an amino acid sequence at least at least 50% (e.g., 50%,60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to the amino acid sequence of SEQ ID NO:7 and a lightchain variable domain having an amino acid sequence at least at least50% (e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:8.

In some embodiments an antigen-binding site of the protein that competeswith the CD33-binding sites includes a heavy chain variable domainhaving an amino acid sequence at least at least 50% (e.g., 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:9 and a light chainvariable domain having an amino acid sequence at least at least 50%(e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10.

In some embodiments an antigen-binding site of the protein that competeswith the CD33-binding sites includes a heavy chain variable domainhaving an amino acid sequence at least at least 50% (e.g., 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:11 and a light chainvariable domain having an amino acid sequence at least at least 50%(e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence of SEQ ID NO:12.

In some embodiments an antigen-binding site of the protein that competeswith the CD33-binding sites includes a heavy chain variable domainhaving an amino acid sequence at least at least 50% (e.g., 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:13 and a light chainvariable domain having an amino acid sequence at least at least 50%(e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence of SEQ ID NO:14.

In some embodiments an antigen-binding site of the protein that competeswith the CD33-binding sites includes a heavy chain variable domainhaving an amino acid sequence at least at least 50% (e.g., 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:15 and a light chainvariable domain having an amino acid sequence at least at least 50%(e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence of SEQ ID NO:16.

In some embodiments an antigen-binding site of the protein that competeswith the CD33-binding sites includes a heavy chain variable domainhaving an amino acid sequence at least at least 50% (e.g., 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:17 and a light chainvariable domain having an amino acid sequence at least at least 50%(e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence of SEQ ID NO:18.

In some embodiments an antigen-binding site of the protein that competeswith the CD33-binding sites includes a heavy chain variable domainhaving an amino acid sequence at least at least 50% (e.g., 50%, 60%,70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:19 and a light chainvariable domain having an amino acid sequence at least at least 50%(e.g., 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence of SEQ ID NO:20.

In certain embodiments, the present invention provides a protein thatincludes an antigen-binding site that competes for binding to human andcynomolgus CD33 with an antibody that includes an antigen-binding sitethat binds a tumor-associated antigen.

In certain embodiments, the present invention provides a protein thatincludes an antigen-binding site that competes for binding to human andcynomolgus CD33 with an antibody that includes an antibody constantregion or a portion thereof capable of binding CD16.

CAR T Cells, CD33/CD3-Directed Bispecific T-Cell Engagers,Immunocytokines, Antibody-Drug Conjugates, and Immunotoxins

Another aspect of the present disclosure provides a molecule or complexcomprising an antigen-binding site that binds CD33 as disclosed herein.Exemplary molecules or complexes include but are not limited to chimericantigen receptors (CARs), T-cell engagers (e.g., CD33/CD3-directedbispecific T-cell engagers), immunocytokines, antibody-drug conjugates,and immunotoxins.

Any antigen-binding site that binds CD33 as disclosed herein can beused, including but not limited to the antigen-binding site that bindsCD33 as disclosed in Section I. Antigen-Binding Site. In certainembodiments, the amino acid sequence(s) of the antigen-binding site thatbinds CD33 are provided in Table 1. In certain embodiments, theantigen-binding site that binds CD33 is an scFv. In certain embodiments,the scFv comprises an amino acid sequence at least 90% (e.g., 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to an aminoacid sequence selected from SEQ ID NO:188, SEQ ID NO:198, SEQ ID NO:206,SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ IDNO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220,SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:447, SEQ IDNO:448, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:451, SEQ ID NO:452, SEQID NO:453, SEQ ID NO:454, SEQ ID NO:455, SEQ ID NO:456, SEQ ID NO:457,SEQ ID NO:458, SEQ ID NO:459, SEQ ID NO:460, SEQ ID NO:461, SEQ IDNO:462, SEQ ID NO:463, SEQ ID NO:464, SEQ ID NO:465, SEQ ID NO:466, SEQID NO:467, SEQ ID NO:468, SEQ ID NO:469, SEQ ID NO:470, SEQ ID NO:471,SEQ ID NO:472, SEQ ID NO:473, SEQ ID NO:474, SEQ ID NO:475, SEQ IDNO:476, SEQ ID NO:477, SEQ ID NO:478, SEQ ID NO:479, SEQ ID NO:480, SEQID NO:481, SEQ ID NO:482, SEQ ID NO:483, and SEQ ID NO:484. In certainembodiments, the scFv comprises an amino acid sequence selected from SEQID NO:188, SEQ ID NO:198, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208,SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ IDNO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222,SEQ ID NO:223, SEQ ID NO:447, SEQ ID NO:448, SEQ ID NO:449, SEQ IDNO:450, SEQ ID NO:451, SEQ ID NO:452, SEQ ID NO:453, SEQ ID NO:454, SEQID NO:455, SEQ ID NO:456, SEQ ID NO:457, SEQ ID NO:458, SEQ ID NO:459,SEQ ID NO:460, SEQ ID NO:461, SEQ ID NO:462, SEQ ID NO:463, SEQ IDNO:464, SEQ ID NO:465, SEQ ID NO:466, SEQ ID NO:467, SEQ ID NO:468, SEQID NO:469, SEQ ID NO:470, SEQ ID NO:471, SEQ ID NO:472, SEQ ID NO:473,SEQ ID NO:474, SEQ ID NO:475, SEQ ID NO:476, SEQ ID NO:477, SEQ IDNO:478, SEQ ID NO:479, SEQ ID NO:480, SEQ ID NO:481, SEQ ID NO:482, SEQID NO:483, and SEQ ID NO:484.

In certain embodiments, the antigen-binding site that binds CD33comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3sequences represented by the amino acid sequences of SEQ ID NOs:181, 46,and 182, respectively; and a light chain variable domain comprisingCDR1, CDR2, and CDR3 sequences represented by the amino acid sequencesof SEQ ID NOs:48, 49, and 50, respectively. In certain embodiments, theantigen-binding site comprises a heavy chain variable domain with anamino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:9; and a light chain variable domain with an amino acid sequence atleast 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:10. In certainembodiments, the antigen-binding site comprises an scFv comprising anamino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:188.

In other embodiments of any one of the foregoing aspects in thissubsection, the antigen-binding site that binds CD33 comprises a heavychain variable domain comprising CDR1, CDR2, and CDR3 sequencesrepresented by the amino acid sequences of SEQ ID NOs:183, 34, and 184,respectively; and a light chain variable domain comprising CDR1, CDR2,and CDR3 sequences represented by the amino acid sequences of SEQ IDNOs:36, 185, and 38, respectively. In certain embodiments, theantigen-binding site comprises a heavy chain variable domain with anamino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:5; and a light chain variable domain with an amino acid sequence atleast 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:6. In certainembodiments, the antigen-binding site comprises an scFv comprising anamino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:198.

Chimeric Antigen Receptors (CARs)

In certain embodiments, the present disclosure provides a CD33-targetingCAR comprising an antigen-binding site that binds CD33 as disclosedherein (see, e.g., Table 1). The CD33-targeting CAR can comprise an Fabfragment or an scFv.

The term “chimeric antigen receptor” or alternatively a “CAR” refers toa recombinant polypeptide construct comprising at least an extracellularantigen binding domain, a transmembrane domain and an intracellularsignaling domain comprising a functional signaling domain derived from astimulatory molecule (also referred to herein as a “primary signalingdomain”).

Accordingly, in certain embodiments, the CAR comprises an extracellularantigen-binding site that binds CD33 as disclosed herein, atransmembrane domain, and an intracellular signaling domain comprising aprimary signaling domain. In certain embodiments, the CAR furthercomprises one or more functional signaling domains derived from at leastone costimulatory molecule (also referred to as a “costimulatorysignaling domain”).

In one embodiment, the CAR comprises a chimeric fusion proteincomprising a CD33-binding domain (e.g., CD33-binding scFv domain)comprising a heavy chain variable domain and a light chain variabledomain listed in Table 1 as an extracellular antigen binding domain, atransmembrane domain, and an intracellular signaling domain comprising aprimary signaling domain. In one embodiment, the CAR comprises achimeric fusion protein comprising a CD33-binding domain (e.g.,CD33-binding scFv domain) comprising a heavy chain variable domain and alight chain variable domain listed in Table 1 as an extracellularantigen binding domain, a transmembrane domain and an intracellularsignaling domain comprising a costimulatory signaling domain and aprimary signaling domain. In one aspect, the CAR comprises a chimericfusion protein comprising a CD33-binding domain (e.g., CD33-binding scFvdomain) comprising a heavy chain variable domain and a light chainvariable domain listed in Table 1 as an extracellular antigen bindingdomain, a transmembrane domain, and an intracellular signaling domaincomprising two costimulatory signaling domains and a primary signalingdomain. In one embodiment, the CAR comprises a chimeric fusion proteincomprising a CD33-binding domain comprising a heavy chain variabledomain and a light chain variable domain listed in Table 1 as anextracellular antigen binding domain, a transmembrane domain, and anintracellular signaling domain comprising at least two costimulatorysignaling domains and a primary signaling domain.

With respect to the transmembrane domain, in various embodiments, theCAR is designed to comprise a transmembrane domain that is fused to theextracellular domain of the CAR. In one embodiment, the transmembranedomain is one that naturally is associated with one of the domains inthe CAR. In some instances, the transmembrane domain can be selected ormodified by amino acid substitution to avoid binding of such domains tothe transmembrane domains of the same or different surface membraneproteins to minimize interactions with other members of the receptorcomplex. In another embodiment, the transmembrane domain is capable ofhomodimerization with another CAR on the CAR T cell surface. In anotherembodiment, the amino acid sequence of the transmembrane domain may bemodified or substituted so as to minimize interactions with the bindingdomains of the native binding partner present in the same CAR T cell.

The transmembrane domain may be derived from any naturally occurringmembrane-bound or transmembrane protein. In one embodiment, thetransmembrane region is capable of signaling to the intracellulardomain(s) whenever the CAR has bound to a target. In some embodiments,the transmembrane domain comprises the transmembrane region(s) of one ormore proteins selected from the group consisting of TCR α chain, TCR βchain, TCR ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22,CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In someembodiments, the transmembrane domain comprises the transmembraneregion(s) of one or more proteins selected from the group consisting ofKIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44,NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a,ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103,ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2,CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84,CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, and NKG2C.

The extracellular CD33-binding domain (e.g., CD33-binding scFv domain)domain can be connected to the transmembrane domain by a hinge region. Avariety of hinges can be employed, including but not limited to thehuman Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), aGly-Ser linker, a (G4S)₄ linker, a KIR2DS2 hinge, and a CD8α hinge.

The intracellular signaling domain of the CAR disclosed herein isresponsible for activation of at least one of the specialized functionsof the immune cell (e.g., cytolytic activity or helper activity,including the secretion of cytokines, of a T cell) in which the CAR hasbeen placed in. Thus, as used herein, the term “intracellular signalingdomain” refers to the portion of a protein which transduces an effectorfunction signal and directs the cell to perform a specialized function.While usually the entire intracellular signaling domain can be employed,in many cases it is not necessary to use the entire chain. To the extentthat a truncated portion of the intracellular signaling domain is used,such truncated portion may be used in place of the intact chain as longas it transduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal.

The intracellular signaling domain of the CAR comprises a primarysignaling domain (i.e., a functional signaling domain derived from astimulatory molecule) and one or more costimulatory signaling domains(i.e., functional signaling domains derived from at least onecostimulatory molecule).

As used herein, the term “stimulatory molecule” refers to a moleculeexpressed by an immune cell, e.g., a T cell, an NK cell, or a B cell,that provide the cytoplasmic signaling sequence(s) that regulateactivation of the immune cell in a stimulatory way for at least someaspect of the immune cell signaling pathway. In one embodiment, thesignal is a primary signal that is initiated by, for instance, bindingof a TCR/CD3 complex with an MEW molecule loaded with a peptide, andwhich leads to mediation of a T cell response, including, but notlimited to, proliferation, activation, differentiation, and the like.

Primary signaling domains that act in a stimulatory manner may containsignaling motifs which are known as immunoreceptor tyrosine-basedactivation motifs or ITAMs. Examples of ITAM containing cytoplasmicsignaling sequences that are of particular use in the present disclosureinclude those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gammaRIIa, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon,CD79a, CD79b, DAP10, and DAP12. In one embodiment, the primary signalingdomain in any one or more CARs disclosed herein comprises a cytoplasmicsignaling sequence derived from CD3-zeta.

In some embodiments, the primary signaling domain is a functionalsignaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta,CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, 4-1BB, and/or CD3-zeta. Inan embodiment, the intracellular signaling domain comprises a functionalsignaling domain of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa,FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a,CD79b, DAP10, and/or DAP12. In a particular embodiment, the primarysignaling domain is a functional signaling domain of the zeta chainassociated with the T cell receptor complex.

As used herein, the term “costimulatory molecule” refers to a cognatebinding partner on a T cell that specifically binds with a costimulatoryligand, thereby mediating a costimulatory response by the T cell, suchas, but not limited to, proliferation. A costimulatory molecule is acell surface molecule other than an antigen receptor or its ligands thatis required for an efficient response of lymphocytes to an antigen.Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40,CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1,CD11a/CD18), CD2, CD7, CD258 (LIGHT), NKG2C, B7-H3, and a ligand thatspecifically binds with CD83, and the like. Further examples of suchcostimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR),SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, and a ligand that specifically binds with CD83. In someembodiments, the costimulatory signaling domain of the CAR is afunctional signaling domain of a costimulatory molecule describedherein, e.g., OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258,NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD11a/CD18),ICOS and 4-1BB (CD137), or any combination thereof.

As used herein, the term “signaling domain” refers to the functionalportion of a protein which acts by transmitting information within thecell to regulate cellular activity via defined signaling pathways bygenerating second messengers or functioning as effectors by respondingto such messengers.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR disclosed herein may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker, for example, between 2 and 10 amino acids in length may form thelinkage.

Another aspect of the present disclosure provides a nucleic acidencoding a CD33-targeting CAR disclosed herein. The nucleic acid isuseful for expressing the CAR in an effector cell (e.g., T cell) byintroducing the nucleic acid to the cell.

Exemplary nucleic acid sequences encoding the extracellular portions ofthe CARs are disclosed herein, e.g., as SEQ ID NOs:246-265.Modifications may be made in the sequence to create an equivalent orimproved variant of the nucleic acid sequences, for example, by changingone or more of the codons according to the codon degeneracy table. A DNAcodon degeneracy table is provided in Table 10.

TABLE 10 Amino Acid Codons One Three letter letter Amino Acids code codeCodons Alanine A Ala GCA GCC GCG GCU Cysteine C Cys UGC UGUAspartic acid D Asp GAC GAU Glutamic acid E Glu GAA GAG Phenylalanine FPhe UUC UUU Glycine G Gly GGA GGC GGG GGU Histidine H His CAC CAUIsoleucine I Iso AUA AUC AUU Lysine K Lys AAA AAG Leucine L LeuUUA UUG CUA CUC CUG CUU Methionine M Met AUG Asparagine N Asn AAC AAUProline P Pro CCA CCC CCG CCU Glutamine Q Gin CAA CAG Arginine R ArgAGA AGG CGA CGC CGG CGU Serine S Ser AGC AGU UCA UCC UCG UCU Threonine TThr ACA ACC ACG ACU Valine V Val GUA GUC GUG GUU Tryptophan W Trp UGGTyrosine Y Tyr UAC UAU

In certain embodiments, the nucleic acid is a DNA molecule (e.g., a cDNAmolecule). In certain embodiments, the nucleic acid further comprises anexpression control sequence (e.g., promoter and/or enhancer) operablylinked to the CAR coding sequence. In certain embodiments, the presentdisclosure provides a vector comprising the nucleic acid. The vector canbe a viral vector (e.g., AAV vector, lentiviral vector, or adenoviralvector) or a non-viral vector (e.g., plasmid).

In certain embodiments, the nucleic acid is an RNA molecule (e.g., anmRNA molecule). A method for generating mRNA for use in transfection caninvolve in vitro transcription of a template with specially designedprimers, followed by polyA addition, to produce an RNA constructcontaining 3′ and 5′ untranslated sequences, a 5′ cap and/or InternalRibosome Entry Site (IRES), the nucleic acid to be expressed, and apolyA tail, typically 50-2000 bases in length. The RNA molecule can befurther modified to increase translational efficiency and/or stability,e.g., as disclosed in U.S. Pat. Nos. 8,278,036; 8,883,506, and8,716,465. RNA molecules so produced can efficiently transfect differentkinds of cells.

In one embodiment, the nucleic acid encodes an amino acid sequencecomprising a signal peptide at the amino-terminus of the CAR. Suchsignal peptide can facilitate the cell surface localization of the CARwhen it is expressed in an effector cell, and is cleaved from the CARduring cellular processing. In one embodiment, the nucleic acid encodesan amino acid sequence comprising a signal peptide at the N-terminus ofthe extracellular CD33-binding domain (e.g., CD33-binding scFv domain).

RNA or DNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation, cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001)).

Another aspect of the present disclosure provides an immune effectorcell expressing the CD33-targeting CAR. Also provided is an immuneeffector cell comprising the nucleic acid encoding the CD33-targetingCAR. The immune effector cells include but are not limited to T cellsand NK cells. In certain embodiments, the T cell is selected from a CD8⁺T cell, a CD4⁺ T cell, and an NKT cell. The T cell or NK cell can be aprimary cell or a cell line.

The immune effector cells can be obtained from a number of sources,including peripheral blood mononuclear cells, bone marrow, lymph nodetissue, cord blood, thymus tissue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors, by methods knownin the art. The immune effector cells can also be differentiated invitro from a pluripotent or multipotent cell (e.g., a hematopoietic stemcell). In some embodiments, the present disclosure provides apluripotent or multipotent cell (e.g., a hematopoietic stem cell)expressing the CD33-targeting CAR or comprising a nucleic acid disclosedherein.

In certain embodiments, the immune effector cells are isolated and/orpurified. For example, regulatory T cells can be removed from a T cellpopulation using a CD25-binding ligand. Effector cells expressing acheckpoint protein (e.g., PD-1, LAG-3, or TIM-3) can be removed bysimilar methods. In certain embodiments, the effector cells are isolatedby a positive selection step. For example, a population of T cells canbe isolated by incubation with anti-CD3/anti-CD28-conjugated beads.Other cell surface markers, such as IFN-7, TNF-α, IL-17A, IL-2, IL-3,IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, can also be usedfor positive selection.

Immune effector cells may be activated and expanded generally usingmethods known in the art, e.g., as described in U.S. Pat. Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent ApplicationPublications Nos. 2006/0121005 and 2016/0340406. For example, in certainembodiments, T cells can be expanded and/or activated by contact with ananti-CD3 antibody and an anti-CD28 antibody, under conditionsappropriate for stimulating proliferation of the T cells. The cells canbe expanded in culture for a period of several hours (e.g., about 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one embodiment, thecells are expanded for a period of 4 to 9 days. Multiple cycles ofstimulation may be desirable for prolonged cell culture (e.g., culturefor a period of 60 days or more). In certain embodiments, the cellculture comprises serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12,IL-15, TGFβ, TNF-α, or a combination thereof. Other additives for thegrowth of cells known to the skilled person, e.g., surfactant,plasmanate, and reducing agents such as N-acetyl-cysteine and2-mercaptoethanol, can also be included in the cell culture. In certainembodiments, the immune effector cell of the present disclosure is acell obtained from in vitro expansion.

Further embodiments of the CD33-targeting CAR (e.g., regulatable CAR),nucleic acid encoding the CAR, and effector cells expressing the CAR orcomprising the nucleic acid are provided in U.S. Pat. Nos. 7,446,190 and9,181,527, U.S. Patent Application Publication Nos. 2016/0340406 and2017/0049819, and International Patent Application Publication No.WO2018/140725.

CD33/CD3-Directed Bispecific T-Cell Engagers

In certain embodiments, the present disclosure provides aCD33/CD3-directed bispecific T-cell engager comprising anantigen-binding site that binds CD33 disclosed herein. In certainembodiments, the CD33/CD3-directed bispecific T-cell engager comprisesan amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:187. In certainembodiments, the CD33/CD3-directed bispecific T-cell engager comprisesan amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:197.

In certain embodiments, the CD33/CD3-directed bispecific T-cell engagerfurther comprises an antigen-binding site that binds CD3. Exemplaryantigen-binding sites that bind CD3 are disclosed in InternationalPatent Application Publication Nos. WO2014/051433 and WO2017/097723.

Another aspect of the present disclosure provides a nucleic acidencoding at least one polypeptide of the CD33/CD3-directed bispecificT-cell engager, wherein the polypeptide comprises an antigen bindingsite that binds CD33. In certain embodiments, the nucleic acid furthercomprises a nucleotide sequence encoding a signal peptide that, whenexpressed, is at the N-terminus of one or more of the polypeptides ofthe CD33/CD3-directed bispecific T-cell engager. Also provided is avector (e.g., a viral vector) comprising the nucleic acid, a producercell comprising the nucleic acid or vector, and a producer cellexpressing the CD33/CD3-directed bispecific T-cell engager.

Immunocytokines

In certain embodiments, the present disclosure provides animmunocytokine comprising an antigen-binding site that binds CD33disclosed herein and a cytokine. Any cytokine (e.g., pro-inflammatorycytokines) known in the art can be used, including but not limited toIL-2, IL-4, IL-10, IL-12, IL-15, TNF, IFNα, IFNγ, and GM-CSF. Moreexemplary cytokines are disclosed in U.S. Pat. No. 9,567,399. In certainembodiments, the antigen-binding site is connected to the cytokine bychemical conjugation (e.g., covalent or noncovalent chemicalconjugation). In certain embodiments, the antigen-binding site isconnected to the cytokine by fusion of polypeptide. The immunocytokinecan further comprise an Fc domain connected to the antigen-binding sitethat binds CD33. In certain embodiments, the immunocytokine comprises anamino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:187. In certainembodiments, the immunocytokine comprises an amino acid sequence atleast 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:197. In certain embodiments, the cytokineis connected to the Fc domain directly or via a linker.

Another aspect of the present disclosure provides a nucleic acidencoding at least one polypeptide of the immunocytokine, wherein thepolypeptide comprises an antigen binding site that binds CD33. Incertain embodiments, the nucleic acid further comprises a nucleotidesequence encoding a signal peptide that, when expressed, is at theN-terminus of one or more of the polypeptides of the immunocytokine.Also provided is a vector (e.g., a viral vector) comprising the nucleicacid, a producer cell comprising the nucleic acid or vector, and aproducer cell expressing the immunocytokine.

Antibody-Drug Conjugates

In certain embodiments, the present disclosure provides an antibody-drugconjugate comprising an antigen-binding site that binds CD33 disclosedherein and a cytotoxic drug moiety. Exemplary cytotoxic drug moietiesare disclosed in International Patent Application Publication Nos.WO2014/160160 and WO2015/143382. In certain embodiments, the cytotoxicdrug moiety is selected from auristatin, N-acetyl-γ calicheamicin,maytansinoid, pyrrolobenzodiazepine, and SN-38. The antigen-binding sitecan be connected to the cytotoxic drug moiety by chemical conjugation(e.g., covalent or noncovalent chemical conjugation). In certainembodiments, the antibody-drug conjugate further comprises an Fc domainconnected to the antigen-binding site that binds CD33. In certainembodiments, the antibody-drug conjugate comprises an amino acidsequence at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to SEQ ID NO:187. In certain embodiments,the antibody-drug conjugate comprises an amino acid sequence at least90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to SEQ ID NO:197. In certain embodiments, the cytotoxic drugmoiety is connected to the Fc domain directly or via a linker.

Immunotoxins

In certain embodiments, the present disclosure provides an immunotoxincomprising an antigen-binding site that binds CD33 disclosed herein anda cytotoxic peptide moiety. Any cytotoxic peptide moiety known in theart can be used, including but not limited to ricin, Diphtheria toxin,and Pseudomonas exotoxin A. More exemplary cytotoxic peptides aredisclosed in International Patent Application Publication Nos.WO2012/154530 and WO2014/164680. In certain embodiments, the cytotoxicpeptide moiety is connected to the protein by chemical conjugation(e.g., covalent or noncovalent chemical conjugation). In certainembodiments, the cytotoxic peptide moiety is connected to the protein byfusion of polypeptide. The immunotoxin can further comprise an Fc domainconnected to the antigen-binding site that binds CD33. In certainembodiments, the immunotoxin comprises an amino acid sequence at least90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to SEQ ID NO:187. In certain embodiments, the immunotoxincomprises an amino acid sequence at least 90% (e.g., 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:197. Incertain embodiments, the cytotoxic peptide moiety is connected to the Fcdomain directly or via a linker.

Another aspect of the present disclosure provides a nucleic acidencoding at least one polypeptide of the immunotoxin, wherein thepolypeptide comprises an antigen binding site that binds CD33. Incertain embodiments, the nucleic acid further comprises a nucleotidesequence encoding a signal peptide that, when expressed, is at theN-terminus of one or more of the polypeptides of the immunotoxin. Alsoprovided is a vector (e.g., a viral vector) comprising the nucleic acid,a producer cell comprising the nucleic acid or vector, and a producercell expressing the immunotoxin.

III. Therapeutic Compositions and their Use

The invention provides methods for treating cancer using amulti-specific binding protein described herein and/or a pharmaceuticalcomposition described herein. The methods may be used to treat a varietyof cancers which express CD33 by administering to a patient in needthereof a therapeutically effective amount of a multi-specific bindingprotein described herein.

The therapeutic method can be characterized according to the cancer tobe treated. For example, in certain embodiments, the cancers are AML,myelodysplastic syndromes, chronic myelomonocytic leukemia, myeloidblast crisis of chronic myeloid leukemia, and ALLs.

For example, in certain embodiments, the cancer is a solid tumor. Incertain other embodiments, the cancer is brain cancer, bladder cancer,breast cancer, cervical cancer, colon cancer, colorectal cancer,endometrial cancer, esophageal cancer, leukemia, lung cancer, livercancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer,rectal cancer, renal cancer, stomach cancer, testicular cancer, oruterine cancer. In yet other embodiments, the cancer is a vascularizedtumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma,melanoma, glioma, neuroblastoma, sarcoma (e.g., an angiosarcoma orchondrosarcoma), larynx cancer, parotid cancer, bilary tract cancer,thyroid cancer, acral lentiginous melanoma, actinic keratoses, acutelymphocytic leukemia, acute myeloid leukemia, adenoid cycstic carcinoma,adenomas, adenosarcoma, adenosquamous carcinoma, anal canal cancer, analcancer, anorectum cancer, astrocytic tumor, bartholin gland carcinoma,basal cell carcinoma, biliary cancer, bone cancer, bone marrow cancer,bronchial cancer, bronchial gland carcinoma, carcinoid,cholangiocarcinoma, chondosarcoma, choriod plexus papilloma/carcinoma,chronic lymphocytic leukemia, chronic myeloid leukemia, clear cellcarcinoma, connective tissue cancer, cystadenoma, digestive systemcancer, duodenum cancer, endocrine system cancer, endodermal sinustumor, endometrial hyperplasia, endometrial stromal sarcoma,endometrioid adenocarcinoma, endothelial cell cancer, ependymal cancer,epithelial cell cancer, Ewing's sarcoma, eye and orbit cancer, femalegenital cancer, focal nodular hyperplasia, gallbladder cancer, gastricantrum cancer, gastric fundus cancer, gastrinoma, glioblastoma,glucagonoma, heart cancer, hemangiblastomas, hemangioendothelioma,hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatobiliarycancer, hepatocellular carcinoma, Hodgkin's disease, ileum cancer,insulinoma, intaepithelial neoplasia, interepithelial squamous cellneoplasia, intrahepatic bile duct cancer, invasive squamous cellcarcinoma, jejunum cancer, joint cancer, Kaposi's sarcoma, pelviccancer, large cell carcinoma, large intestine cancer, leiomyosarcoma,lentigo maligna melanomas, lymphoma, male genital cancer, malignantmelanoma, malignant mesothelial tumors, medulloblastoma,medulloepithelioma, meningeal cancer, mesothelial cancer, metastaticcarcinoma, mouth cancer, mucoepidermoid carcinoma, multiple myeloma,muscle cancer, nasal tract cancer, nervous system cancer,neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skincancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglialcancer, oral cavity cancer, osteosarcoma, papillary serousadenocarcinoma, penile cancer, pharynx cancer, pituitary tumors,plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renalcell carcinoma, respiratory system cancer, retinoblastoma,rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer,small cell carcinoma, small intestine cancer, smooth muscle cancer, softtissue cancer, somatostatin-secreting tumor, spine cancer, squamous cellcarcinoma, striated muscle cancer, submesothelial cancer, superficialspreading melanoma, T cell leukemia, tongue cancer, undifferentiatedcarcinoma, ureter cancer, urethra cancer, urinary bladder cancer,urinary system cancer, uterine cervix cancer, uterine corpus cancer,uveal melanoma, vaginal cancer, verrucous carcinoma, VIPoma, vulvacancer, well differentiated carcinoma, or Wilms tumor.

In certain other embodiments, the cancer is non-Hodgkin's lymphoma, suchas a B-cell lymphoma or a T-cell lymphoma. In certain embodiments, thenon-Hodgkin's lymphoma is a B-cell lymphoma, such as a diffuse largeB-cell lymphoma, primary mediastinal B-cell lymphoma, follicularlymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginalzone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma,Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, orprimary central nervous system (CNS) lymphoma. In certain otherembodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, such as aprecursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma,cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma,extranodal natural killer/T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma, or peripheral T-cell lymphoma.

The cancer to be treated can be characterized according to the presenceof a particular antigen expressed on the surface of the cancer cell. Incertain embodiments, the cancer cell can express one or more of thefollowing in addition to CD33: CD2, CD19, CD20, CD30, CD38, CD40, CD52,CD70, EGFR/ERBB1, IGF1R, HER3/ERBB3, HER4/ERBB4, MUC1, TROP2, cMET,SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4,and PD1.

In embodiments of the present invention, the cancer to be treated isselected from acute myeloid leukemia (AML), myelodysplastic syndrome(MDS), acute lymphoblastic leukemia (ALL), myeloproliferative neoplasms(MPNs), lymphoma, non-Hodgkin lymphomas, and classical Hodgkin lymphoma.

In some embodiments of the present invention, the cancer to be treatedis AML. In some embodiments of the present invention, the AML isselected from undifferentiated acute myeloblastic leukemia, acutemyeloblastic leukemia with minimal maturation, acute myeloblasticleukemia with maturation, acute promyelocytic leukemia (APL), acutemyelomonocytic leukemia, acute myelomonocytic leukemia witheosinophilia, acute monocytic leukemia, acute erythroid leukemia, acutemegakaryoblastic leukemia (AMKL), acute basophilic leukemia, acutepanmyelosis with fibrosis, and blastic plasmacytoid dendritic cellneoplasm (BPDCN). In some embodiments of the present invention, the AMLis characterized by expression of CLL-1 on the AML leukemia stem cells(LSCs). In some embodiments of the present invention, the LSCs in an AMLsubject further express a membrane marker selected from CD34, CD38,CD123, TIM3, CD25, CD32, and CD96. In some embodiments of the presentinvention, the AML is characterized as a minimal residual disease (MRD).In some embodiments of the present invention, the MRD of AML ischaracterized by the presence or absence of a mutation selected fromFLT3-ITD ((Fms-like tyrosine kinase 3)-internal tandem duplications(ITD)), NPM1 (Nucleophosmin 1), DNMT3A (DNA methyltransferase geneDNMT3A), and IDH (Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2)).

In certain embodiments of the present invention, the cancer is MDSselected from MDS with multilineage dysplasia (MDS-MLD), MDS with singlelineage dysplasia (MDS-SLD), MDS with ring sideroblasts (MDS-RS), MDSwith excess blasts (MDS-EB), MDS with isolated del(5q), and MDS,unclassified (MDS-U).

It is contemplated that the multi-specific binding proteins and/orpharmaceutical compositions of the present disclosure can be used totreat a variety of cancers, not limited to cancers in which the cancercells express CD33. For example, in certain embodiments, themulti-specific binding proteins and/or pharmaceutical compositionsdisclosed herein can be used to treat cancers that are associated withCD33-expressing immune cells. CD33 is expressed on many myeloidlineages, and tumor-infiltrating myeloid cells (e.g., tumor-associatedmacrophages) may contribute to cancer progression and metastasis.Therefore, the methods disclosed herein may be used to treat a varietyof cancers in which CD33 is expressed, whether on cancer cells or onimmune cells.

In certain embodiments, the multi-specific binding proteins and/orpharmaceutical compositions of the present disclosure can be used totreat cancers that express an Fc receptor with a higher binding affinityto Fc (e.g., IgG1 Fc) than CD16. In certain embodiments, the Fc receptoris FcγRI. In certain embodiments, the Fc receptor is expressed on cancercells and/or other cells in the tumor microenvironment.

In certain embodiments, the patient has effector cells (e.g., NK cells)that express a CD16 variant with V158F substitution. In certainembodiments, the patient has a single nucleotide polymorphism (SNP) inthe CD16 gene that causes V158F substitution. In certain embodiments,the patient has such an SNP in only one allele. In certain embodiments,the patient has such an SNP or SNPs in both alleles.

IV. Combination Therapy

Another aspect of the invention provides for combination therapy.Multi-specific binding proteins described herein be used in combinationwith additional therapeutic agents to treat the cancer.

Exemplary therapeutic agents that may be used as part of a combinationtherapy in treating cancer, include, for example, radiation, mitomycin,tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine,mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin,nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed,daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane,nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone,aminoglutethimide, amsacrine, proglumide, elliptinium acetate,ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin,nimustine, vindesine, flutamide, drogenil, butocin, carmofur, razoxane,sizofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine,picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride,oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol,formestane, interferon-alpha, interferon-2 alpha, interferon-beta,interferon-gamma, colony stimulating factor-1, colony stimulatingfactor-2, denileukin diftitox, interleukin-2, luteinizing hormonereleasing factor and variations of the aforementioned agents that mayexhibit differential binding to its cognate receptor, and increased ordecreased serum half-life.

An additional class of agents that may be used as part of a combinationtherapy in treating cancer is immune checkpoint inhibitors. Exemplaryimmune checkpoint inhibitors include agents that inhibit one or more of(i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmedcell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi)B7-H4, and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approvedby the United States Food and Drug Administration for treating melanoma.

Yet other agents that may be used as part of a combination therapy intreating cancer are monoclonal antibody agents that targetnon-checkpoint targets (e.g., herceptin) and non-cytotoxic agents (e.g.,tyrosine-kinase inhibitors).

Yet other categories of anti-cancer agents include, for example: (i) aninhibitor selected from an ALK Inhibitor, an ATR Inhibitor, an A2AAntagonist, a Base Excision Repair Inhibitor, a Bcr-Abl Tyrosine KinaseInhibitor, a Bruton's Tyrosine Kinase Inhibitor, a CDC7 Inhibitor, aCHK1 Inhibitor, a Cyclin-Dependent Kinase Inhibitor, a DNA-PK Inhibitor,an Inhibitor of both DNA-PK and mTOR, a DNMT1 Inhibitor, a DNMT1Inhibitor plus 2-chloro-deoxyadenosine, an HDAC Inhibitor, a HedgehogSignaling Pathway Inhibitor, an IDO Inhibitor, a JAK Inhibitor, a mTORInhibitor, a MEK Inhibitor, a MELK Inhibitor, a MTH1 Inhibitor, a PARPInhibitor, a Phosphoinositide 3-Kinase Inhibitor, an Inhibitor of bothPARP1 and DHODH, a Proteasome Inhibitor, a Topoisomerase-II Inhibitor, aTyrosine Kinase Inhibitor, a VEGFR Inhibitor, and a WEE1 Inhibitor; (ii)an agonist of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS;and (iii) a cytokine selected from IL-12, IL-15, GM-CSF, and G-CSF.

Proteins of the invention can also be used as an adjunct to surgicalremoval of the primary lesion.

The amount of multi-specific binding protein and additional therapeuticagent and the relative timing of administration may be selected in orderto achieve a desired combined therapeutic effect. For example, whenadministering a combination therapy to a patient in need of suchadministration, the therapeutic agents in the combination, or apharmaceutical composition or compositions comprising the therapeuticagents, may be administered in any order such as, for example,sequentially, concurrently, together, simultaneously and the like.Further, for example, a multi-specific binding protein may beadministered during a time when the additional therapeutic agent(s)exerts its prophylactic or therapeutic effect, or vice versa.

V. Pharmaceutical Compositions

The present disclosure also features pharmaceutical compositions thatcontain a therapeutically effective amount of a protein describedherein. The composition can be formulated for use in a variety of drugdelivery systems. One or more physiologically acceptable excipients orcarriers can also be included in the composition for proper formulation.Suitable formulations for use in the present disclosure are found inRemington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed., 1985. For a brief review of methods fordrug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).

In one aspect, the present disclosure provides a formulation of aprotein, which contains a CD33-binding site described herein, and apharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition includes aprotein that includes an antigen-binding site with a heavy chainvariable domain having an amino acid sequence at least 90% (e.g., 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the aminoacid sequence of SEQ ID NO:1, and a light chain variable domain havingan amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ IDNO:2. In certain embodiments, the formulation includes a protein thatincludes an antigen-binding site with a heavy chain variable domainhaving an amino acid sequence at least 90% (e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequenceof SEQ ID NO:3, and a light chain variable domain having an amino acidsequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%) identical to the amino acid sequence of SEQ ID NO:4. Incertain embodiments, the formulation includes a protein that includes anantigen-binding site with a heavy chain variable domain having an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:5,and a light chain variable domain having an amino acid sequence at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:6. In certainembodiments, the formulation includes a protein that includes anantigen-binding site with a heavy chain variable domain having an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:7,and a light chain variable domain having an amino acid sequence at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:8. In certainembodiments, the formulation includes a protein that includes anantigen-binding site with a heavy chain variable domain having an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:9,and a light chain variable domain having an amino acid sequence at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:10. In certainembodiments, the formulation includes a protein that includes anantigen-binding site with a heavy chain variable domain having an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11,and a light chain variable domain having an amino acid sequence at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:12. In certainembodiments, the formulation includes a protein that includes anantigen-binding site with a heavy chain variable domain having an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:13,and a light chain variable domain having an amino acid sequence at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:14. In certainembodiments, the formulation includes a protein that includes anantigen-binding site with a heavy chain variable domain having an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:15,and a light chain variable domain having an amino acid sequence at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:16. In certainembodiments, the formulation includes a protein that includes anantigen-binding site with a heavy chain variable domain having an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:17,and a light chain variable domain having an amino acid sequence at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:18. In certainembodiments, the formulation includes a protein that includes anantigen-binding site with a heavy chain variable domain having an aminoacid sequence at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:19,and a light chain variable domain having an amino acid sequence at least90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the amino acid sequence of SEQ ID NO:20.

The composition can be formulated for use in a variety of drug deliverysystems. One or more physiologically acceptable excipients or carrierscan be included in the composition for proper formulation. Suitableformulations for use in the present disclosure are found in Remington'sPharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.,17th ed., 1985. For a brief review of methods for drug delivery, see,e.g., Langer (Science 249:1527-1533, 1990).

For example, this present disclosure could exist in an aqueouspharmaceutical formulation including a therapeutically effective amountof the protein in a buffered solution forming a formulation. Aqueouscarriers can include sterile water for injection (SWFI), bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution. In certain embodiments, an aqueous formulation isprepared including the protein disclosed herein in a pH-bufferedsolution. The pH of the preparations typically will be between 3 and 11,more preferably between 5 and 9 or between 6 and 8, and most preferablybetween 7 and 8, such as 7 to 7.5. Ranges intermediate to the aboverecited pH's are also intended to be part of this disclosure. Forexample, ranges of values using a combination of any of the aboverecited values as upper and/or lower limits are intended to be included.Examples of buffers that will control the pH within this range includeacetate (e.g., sodium acetate), succinate (such as sodium succinate),gluconate, histidine, citrate and other organic acid buffers. In certainembodiments, the buffer system includes citric acid monohydrate, sodiumcitrate, disodium phosphate dihydrate, and/or sodium dihydrogenphosphate dihydrate. In certain embodiments, the buffer system includesabout 1.3 mg/mL of citric acid (e.g., 1.305 mg/mL), about 0.3 mg/mL ofsodium citrate (e.g., 0.305 mg/mL), about 1.5 mg/mL of disodiumphosphate dihydrate (e.g. 1.53 mg/mL), about 0.9 mg/mL of sodiumdihydrogen phosphate dihydrate (e.g., 0.86), and about 6.2 mg/mL ofsodium chloride (e.g., 6.165 mg/mL). In certain embodiments, the buffersystem includes 1-1.5 mg/mL of citric acid, 0.25 to 0.5 mg/mL of sodiumcitrate, 1.25 to 1.75 mg/ml of disodium phosphate dihydrate, 0.7 to 1.1mg/mL of sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4 mg/mL ofsodium chloride. The pH of the liquid formulation may be set by additionof a pharmaceutically acceptable acid and/or base. In certainembodiments, the pharmaceutically acceptable acid may be hydrochloricacid. In certain embodiments, the base may be sodium hydroxide.

In some embodiments, the formulation include an aqueous carrier, whichis pharmaceutically acceptable (safe and non-toxic for administration toa human) and is useful for the preparation of a liquid formulation.Illustrative carriers include sterile water for injection (SWFI),bacteriostatic water for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution.

A polyol, which acts as a tonicifier and may stabilize the antibody, mayalso be included in the formulation. The polyol is added to theformulation in an amount which may vary with respect to the desiredisotonicity of the formulation. In certain embodiments, the aqueousformulation may be isotonic. The amount of polyol added may also bealtered with respect to the molecular weight of the polyol. For example,a lower amount of a monosaccharide (e.g., mannitol) may be added,compared to a disaccharide (such as trehalose). In certain embodiments,the polyol which may be used in the formulation as a tonicity agent ismannitol. In certain embodiments, the mannitol concentration may beabout 5 to about 20 mg/mL. In certain embodiments, the concentration ofmannitol may be about 7.5 to 15 mg/mL. In certain embodiments, theconcentration of mannitol may be about 10-14 mg/mL. In certainembodiments, the concentration of mannitol may be about 12 mg/mL. Incertain embodiments, the polyol sorbitol may be included in theformulation.

A detergent or surfactant may also be added to the formulation.Exemplary detergents include nonionic detergents such as polysorbates(e.g., polysorbates 20, 80 etc.) or poloxamers (e.g., poloxamer 188).The amount of detergent added is such that it reduces aggregation of theformulated antibody and/or minimizes the formation of particulates inthe formulation and/or reduces adsorption. In certain embodiments, theformulation may include a surfactant which is a polysorbate. In certainembodiments, the formulation may contain the detergent polysorbate 80 orTween 80. Tween 80 is a term used to describe polyoxyethylene (20)sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio CantorVerlag Aulendorf, 4th edi., 1996). In certain embodiments, theformulation may contain between about 0.1 mg/mL and about 10 mg/mL ofpolysorbate 80, or between about 0.5 mg/mL and about 5 mg/mL. In certainembodiments, about 0.1% polysorbate 80 may be added in the formulation.

In certain embodiments, the liquid formulation of the disclosure may beprepared as a 10 mg/mL concentration solution in combination with asugar at stabilizing levels. In certain embodiments the liquidformulation may be prepared in an aqueous carrier. In certainembodiments, a stabilizer may be added in an amount no greater than thatwhich may result in a viscosity undesirable or unsuitable forintravenous administration. In certain embodiments, the sugar may bedisaccharides, e.g., sucrose. In certain embodiments, the liquidformulation may also include one or more of a buffering agent, asurfactant, and a preservative, which is added to the formulationsherein to reduce bacterial action. The addition of a preservative may,for example, facilitate the production of a multi-use (multiple-dose)formulation.

In some embodiments, the present disclosure provides a formulation withan extended shelf life including the protein of the present disclosure,in combination with mannitol, citric acid monohydrate, sodium citrate,disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate,sodium chloride, polysorbate 80, water, and sodium hydroxide.

Deamidation is a common product variant of peptides and proteins thatmay occur during fermentation, harvest/cell clarification, purification,drug substance/drug product storage and during sample analysis.Deamidation is the loss of NH3 from a protein forming a succinimideintermediate that can undergo hydrolysis. The succinimide intermediateresults in a 17 u mass decrease of the parent peptide. The subsequenthydrolysis results in an 18 u mass increase. Isolation of thesuccinimide intermediate is difficult due to instability under aqueousconditions. As such, deamidation is typically detectable as 1 u massincrease. Deamidation of an asparagine results in either aspartic orisoaspartic acid. The parameters affecting the rate of deamidationinclude pH, temperature, solvent dielectric constant, ionic strength,primary sequence, local polypeptide conformation and tertiary structure.The amino acid residues adjacent to Asn in the peptide chain affectdeamidation rates. Gly and Ser following an Asn in protein sequencesresults in a higher susceptibility to deamidation. In certainembodiments, the liquid formulation of the present disclosure may bepreserved under conditions of pH and humidity to prevent deamination ofthe protein product.

In some embodiment, the formulation is a lyophilized formulation. Incertain embodiments, the formulation is freeze-dried (lyophilized) andcontained in about 12-60 vials. In certain embodiments, the formulationis freeze-dried and 45 mg of the freeze-dried formulation may becontained in one vial. In certain embodiments, the about 40 mg-about 100mg of freeze-dried formulation is contained in one vial. In certainembodiments, freeze dried formulation from 12, 27, or 45 vials arecombined to obtained a therapeutic dose of the protein in theintravenous drug formulation. The formulation may be a liquidformulation. In some embodiments, a liquid formulation is stored asabout 250 mg/vial to about 1000 mg/vial. In certain embodiments, theliquid formulation is stored as about 600 mg/vial. In certainembodiments, the liquid formulation is stored as about 250 mg/vial.

In some embodiments, the lyophilized formulation includes the proteinsdescribed herein and a lyoprotectant. The lyoprotectant may be sugar,e.g., disaccharides. In certain embodiments, the lyoprotectant may besucrose or maltose. The lyophilized formulation may also include one ormore of a buffering agent, a surfactant, a bulking agent, and/or apreservative. The amount of sucrose or maltose useful for stabilizationof the lyophilized drug product may be in a weight ratio of at least 1:2protein to sucrose or maltose. In certain embodiments, the protein tosucrose or maltose weight ratio may be of from 1:2 to 1:5.

In certain embodiments, the pH of the formulation, prior tolyophilization, may be set by addition of a pharmaceutically acceptableacid and/or base. In certain embodiments the pharmaceutically acceptableacid may be hydrochloric acid. In certain embodiments, thepharmaceutically acceptable base may be sodium hydroxide. Beforelyophilization, the pH of the solution containing the protein of thepresent disclosure may be adjusted between 6 to 8. In certainembodiments, the pH range for the lyophilized drug product may be from 7to 8.

In certain embodiments, a “bulking agent” may be added. A “bulkingagent” is a compound which adds mass to a lyophilized mixture andcontributes to the physical structure of the lyophilized cake (e.g.,facilitates the production of an essentially uniform lyophilized cakewhich maintains an open pore structure). Illustrative bulking agentsinclude mannitol, glycine, polyethylene glycol and sorbitol. Thelyophilized formulations of the present invention may contain suchbulking agents.

In certain embodiments, the lyophilized protein product is constitutedwith an aqueous carrier. The aqueous carrier of interest herein is onewhich is pharmaceutically acceptable (e.g., safe and non-toxic foradministration to a human) and is useful for the preparation of a liquidformulation, after lyophilization. Illustrative diluents include sterilewater for injection (SWFI), bacteriostatic water for injection (BWFI), apH buffered solution (e.g., phosphate-buffered saline), sterile salinesolution, Ringer's solution or dextrose solution. In certainembodiments, the lyophilized drug product of the current disclosure isreconstituted with either Sterile Water for Injection, USP (SWFI) or0.9% Sodium Chloride Injection, USP. During reconstitution, thelyophilized powder dissolves into a solution. In certain embodiments,the lyophilized protein product of the instant disclosure is constitutedto about 4.5 mL water for injection and diluted with 0.9% salinesolution (sodium chloride solution).

The protein compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as-is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The resulting compositions in solid form may be packagedin multiple single dose units, each containing a fixed amount of theabove-mentioned agent or agents. The composition in solid form can alsobe packaged in a container for a flexible quantity.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The specific dose can be a uniform dose for each patient, for example,50-5000 mg of protein. Alternatively, a patient's dose can be tailoredto the approximate body weight or surface area of the patient. Otherfactors in determining the appropriate dosage can include the disease orcondition to be treated or prevented, the severity of the disease, theroute of administration, and the age, sex and medical condition of thepatient. Further refinement of the calculations necessary to determinethe appropriate dosage for treatment is routinely made by those skilledin the art, especially in light of the dosage information and assaysdisclosed herein. The dosage can also be determined through the use ofknown assays for determining dosages used in conjunction withappropriate dose-response data. An individual patient's dosage can beadjusted as the progress of the disease is monitored. Blood levels ofthe targetable construct or complex in a patient can be measured to seeif the dosage needs to be adjusted to reach or maintain an effectiveconcentration. Pharmacogenomics may be used to determine whichtargetable constructs and/or complexes, and dosages thereof, are mostlikely to be effective for a given individual (Schmitz et al., Clinica.Chimica. Acta. 308: 43-53, 2001; Steimer et al., Clinica. Chimica. Acta.308: 33-41, 2001).

In general, dosages based on body weight are from about 0.01 m to about100 mg per kg of body weight, such as about 0.01 m to about 100 mg/kg ofbody weight, about 0.01 μg to about 50 mg/kg of body weight, about 0.01m to about 10 mg/kg of body weight, about 0.01 m to about 1 mg/kg ofbody weight, about 0.01 m to about 100 μg/kg of body weight, about 0.01m to about 50 μg/kg of body weight, about 0.01 m to about 10 μg/kg ofbody weight, about 0.01 m to about 1 μg/kg of body weight, about 0.01 mto about 0.1 μg/kg of body weight, about 0.1 m to about 100 mg/kg ofbody weight, about 0.1 m to about 50 mg/kg of body weight, about 0.1 mto about 10 mg/kg of body weight, about 0.1 m to about 1 mg/kg of bodyweight, about 0.1 m to about 100 μg/kg of body weight, about 0.1 m toabout 10 μg/kg of body weight, about 0.1 m to about 1 μg/kg of bodyweight, about 1 μg to about 100 mg/kg of body weight, about 1 μg toabout 50 mg/kg of body weight, about 1 μg to about 10 mg/kg of bodyweight, about 1 μg to about 1 mg/kg of body weight, about 1 μg to about100 μg/kg of body weight, about 1 μg to about 50 μg/kg of body weight,about 1 μg to about 10 μg/kg of body weight, about 10 μg to about 100mg/kg of body weight, about 10 μg to about 50 mg/kg of body weight,about 10 μg to about 10 mg/kg of body weight, about 10 μg to about 1mg/kg of body weight, about 10 μg to about 100 μg/kg of body weight,about 10 μg to about 50 μg/kg of body weight, about 50 μg to about 100mg/kg of body weight, about 50 μg to about 50 mg/kg of body weight,about 50 μg to about 10 mg/kg of body weight, about 50 μg to about 1mg/kg of body weight, about 50 μg to about 100 μg/kg of body weight,about 100 m to about 100 mg/kg of body weight, about 100 m to about 50mg/kg of body weight, about 100 m to about 10 mg/kg of body weight,about 100 m to about 1 mg/kg of body weight, about 1 mg to about 100mg/kg of body weight, about 1 mg to about 50 mg/kg of body weight, about1 mg to about 10 mg/kg of body weight, about 10 mg to about 100 mg/kg ofbody weight, about 10 mg to about 50 mg/kg of body weight, about 50 mgto about 100 mg/kg of body weight. Doses may be given once or more timesdaily, weekly, monthly or yearly, or even once every 2 to 20 years.Persons of ordinary skill in the art can easily estimate repetitionrates for dosing based on measured residence times and concentrations ofthe targetable construct or complex in bodily fluids or tissues.Administration of the present invention could be intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous,intrapleural, intrathecal, intracavitary, by perfusion through acatheter or by direct intralesional injection. This may be administeredonce or more times daily, once or more times weekly, once or more timesmonthly, and once or more times annually.

The description above describes multiple aspects and embodiments of theinvention. The patent application specifically contemplates allcombinations and permutations of the aspects and embodiments.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes andmethods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions ofthe present invention that consist essentially of, or consist of, therecited components, and that there are processes and methods accordingto the present invention that consist essentially of, or consist of, therecited processing steps.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components.

Further, it should be understood that elements and/or features of acomposition or a method described herein can be combined in a variety ofways without departing from the spirit and scope of the presentinvention, whether explicit or implicit herein. For example, wherereference is made to a particular compound, that compound can be used invarious embodiments of compositions of the present invention and/or inmethods of the present invention, unless otherwise understood from thecontext. In other words, within this application, embodiments have beendescribed and depicted in a way that enables a clear and conciseapplication to be written and drawn, but it is intended and will beappreciated that embodiments may be variously combined or separatedwithout parting from the present teachings and invention(s). Forexample, it will be appreciated that all features described and depictedherein can be applicable to all aspects of the invention(s) describedand depicted herein.

It should be understood that the expression “at least one of” includesindividually each of the recited objects after the expression and thevarious combinations of two or more of the recited objects unlessotherwise understood from the context and use. The expression “and/or”in connection with three or more recited objects should be understood tohave the same meaning unless otherwise understood from the context.

The use of the term “include,” “includes,” “including,” “have,” “has,”“having,” “contain,” “contains,” or “containing,” including grammaticalequivalents thereof, should be understood generally as open-ended andnon-limiting, for example, not excluding additional unrecited elementsor steps, unless otherwise specifically stated or understood from thecontext.

Where the use of the term “about” is before a quantitative value, thepresent invention also includes the specific quantitative value itself,unless specifically stated otherwise. As used herein, the term “about”refers to a ±10% variation from the nominal value unless otherwiseindicated or inferred.

It should be understood that the order of steps or order for performingcertain actions is immaterial so long as the present invention remainoperable. Moreover, two or more steps or actions may be conductedsimultaneously.

The use of any and all examples, or exemplary language herein, forexample, “such as” or “including,” is intended merely to illustratebetter the present invention and does not pose a limitation on the scopeof the invention unless claimed. No language in the specification shouldbe construed as indicating any non-claimed element as essential to thepractice of the present invention.

EXAMPLES

The following examples are merely illustrative and are not intended tolimit the scope or content of the invention in any way.

Example 1. Kinetics and Affinity of Binding to Different Variants ofCD33

Kinetics and affinity of a series of Fab fragments of anti-CD33antibodies with different CD33 variants (human CD33 ECD, cyno CD33 ECD,V-domain of human CD33, C-domain or human CD33 and selected CD33 SNPs)were assessed by surface plasmon resonance using Biacore 8K instrument(GE Healthcare). Anti-Fab antibody was immobilized on a CMS chip usingstandard amine coupling chemistry. CD33 FABs were captured on theanti-Fab chip at a density of ˜100 RU. Solutions containing differentconcentrations of soluble monomeric CD33 or its domains were injectedover the captured FABs and control surfaces at 30 μl/min at 37° C.Surfaces were regenerated between cycles by quick injection of 10 mMglycine, pH 1.8. To obtain kinetic rate constants double-referenced datawere fit to a 1:1 interaction model using Biacore 8K Evaluation software(GE Healthcare). The equilibrium binding constant K_(D) was determinedby the ratio of binding rate constants k_(d)/k_(a).

Octet Platform-Based Kinetic and Affinity Analysis

ForteBio affinity measurements were performed on an Octet HTX generallyas described in Estep et al., High throughput solution-based measurementof antibody-antigen affinity and epitope binning. Mabs 5(2), 270-278(2013). Briefly, ForteBio affinity measurements were performed byloading IgGs on-line onto AHC sensors. Sensors were equilibratedoff-line in assay buffer for 30 min and then monitored on-line for 60seconds for baseline establishment. Sensors with loaded IgGs wereexposed to 100 nM antigen for 3 minutes, and afterwards were transferredto assay buffer for 3 min for off-rate measurement. All kinetics wereanalyzed using the 1:1 binding model. The results are shown in Table 11.

TABLE 11 Kinetic parameters of human CD33 binding to IgG antibodiesmeasured by BLI. IgG ADI Name K_(D) (M) k_(on) (1/M/s) k_(off) (1/s)ADI-10154 3.70E−09 2.46E+05 8.97E−04 ADI-10155 5.30E−09 2.29E+051.21E−03 ADI-10157 4.20E−09 7.99E+04 3.36E−04 ADI-10158 2.60E−095.86E+05 1.55E−03 ADI-10159 5.80E−10 3.48E+05 2.00E−04 ADI-101605.10E−09 6.38E+05 3.25E−03 ADI-10161 3.70E−09 5.87E+05 2.17E−03ADI-10163 6.80E−09 6.65E+05 4.53E−03 ADI-10164 4.30E−09 3.44E+051.48E−03 ADI-10165 4.00E−09 7.79E+05 3.11E−03 ADI-10167 3.90E−091.00E+06 3.93E−03 ADI-10168 1.00E−08 4.76E+05 4.78E−03 ADI-101739.70E−09 3.24E+05 3.14E−03 ADI-10177 3.60E−09 6.01E+05 2.14E−03ADI-11776 2.94E−10 6.79E+05 2.00E−04 ADI-11801 4.57E−10 4.38E+052.00E−04 ADI-11802 4.53E−10 4.41E+05 2.00E−04 ADI-11807 3.07E−106.51E+05 2.00E−04 ADI-11809 3.13E−10 6.38E+05 2.00E−04 ADI-118121.91E−09 1.66E+05 3.15E−04 ADI-11815 1.44E−09 1.59E+05 2.29E−04ADI-11819 2.43E−10 8.24E+05 2.00E−04 ADI-11825 5.40E−10 6.96E+053.76E−04 ADI-11826 3.93E−10 6.19E+05 2.43E−04 ADI-11828 8.10E−106.58E+05 5.33E−04 ADI-11830 1.20E−09 7.70E+05 9.23E−04 ADI-118352.81E−10 8.44E+05 2.37E−04 ADI-11839 2.45E−09 7.04E+05 1.72E−03Lintuzumab 2.21E−09 4.31E+05 9.52E−04

Example 2. CD33 Antibodies Bind to Human CD33 with High Affinity andCross-React with Cyno CD33

Despite rather high homology between human and cyno CD33 (87% in theECD), most of commercially available anti-CD33 antibodies, e.g.,lintuzumab, mylotarg, etc. lack cross-reactivity with cyno CD33. FIG. 2shows alignment of full length human and cyno CD33 highlighting thedifferences in the primary sequence in the ECD domain.

Affinity of 29 Fab fragments binding to human and cyno CD33 ECD wereassayed by Biacore analysis. Eight out of 29 antibodies showcross-reactivity with cyno CD33. Kinetic parameters of binding are givenin Table 12. Data are compared to lintuzumab. Several antibodies showaffinities >100 fold higher than lintuzumab.

Binding of the Fab fragments from CD33 monoclonal antibodies to thehuman CD33 extracellular domain (ECD) was measured by Biacore at 37° C.The Biacore profile of ADI-10159 is shown in FIG. 3A; the Biacoreprofile of ADI-10177 is shown in FIG. 3B; the Biacore profile ofADI-11776 is shown in FIG. 3C; the Biacore profile of ADI-11801 in FIG.3D; the Biacore profile of ADI-11807 is shown in FIG. 3E; the Biacoreprofile of ADI-11809 is shown in FIG. 3F; the Biacore profile ofADI-11815 is shown in FIG. 3G; the Biacore profile of ADI-11819 FIG. 3H;the Biacore profile of ADI-11830 is shown in FIG. 3I; the Biacoreprofile of ADI-11835 is shown in FIG. 3J; and the Biacore profile of theFab fragment from Lintuzumab is shown in FIG. FIG. 3K.

Binding of the Fab fragments from CD33 monoclonal antibodies to the cynoCD33 ECD was measured by Biacore at 37° C. The Biacore profile ofADI-10159 is shown in FIG. 4A; the Biacore profile of ADI-10177 in FIG.4B; the Biacore profile of ADI-11776 is shown in FIG. 4C; the Biacoreprofile of ADI11807 is shown in FIG. 4D; the Biacore profile ofADI-11809 is shown in FIG. 4E; the Biacore profile of ADI-11819 is shownin FIG. 4F; the Biacore profile of ADI-11830 is shown in FIG. 4G; andthe Biacore profile of ADI-11835 is shown in FIG. 4H.

Binding of the Fab fragments from CD33 monoclonal antibodies to V domainand C domain of human CD33 was measured by Biacore at 37° C. FIGS. 5A-5Jrepresent binding to the V-domain; panels K-T represent binding to the Cdomain. Both FIGS. 5A and 5K are Biacore profiles of ADI-10159; bothFIGS. 5B and 5L are Biacore profiles of ADI-10177; both FIGS. 5C and 5Mare Biacore profiles of ADI-11776; both FIGS. 5D and 5N are Biacoreprofiles of ADI-11801; both FIGS. 5E and 5O are Biacore profiles ofADI-11807; both FIGS. 5F and 5P are Biacore profiles of ADI-11809; bothFIGS. 5G and 5Q are Biacore profiles of ADI-11815; both FIGS. 5H and 5Rare Biacore profiles of ADI-11819; both FIGS. 5I and 5S are Biacoreprofiles of ADI-11830; and FIGS. 5J and 5T are Biacore profiles ofADI-11835.

TABLE 12 Kinetic parameters of human CD33 ECD and cyno CD33 ECD bindingto Fabs measured by SPR at 37° C. No binding is defined as absence ofsignal at highest concentration of 100 nM. Human CD33 Cyno CD33 k_(a)k_(a) Antibody (1/Ms) k_(d) (1/s) K_(D) (nM) (1/Ms) k_(d) (1/s) K_(D)(nM) ADI-10159 4.29e5 1.95e−3 4.53 5.28e5 4.36e−2 74.9 ADI-10177 5.58e56.36e−3 11.4 3.53e5 7.54e−3 21.4 ADI-11776 1.6e6  1.24e−3 0.78 2.62e64.72e−3 1.81 ADI-11807 1.14e6 3.32e−4 0.29 9.98e5 1.51e−4 0.15 ADI-118091.02e6 1.23e−6 0.0012 1.06e6 1.26e−4 0.118 ADI-11819 2.84e6 5.65e−5 0.023.82e6 1.90e−2 4.96 ADI-11830 1.99e6 5.47e−3 2.75 1.34e6 3.64e−2 27.8ADI-11835 2.6e6  1.43e−3 0.53 2.76e6 1.89e−2 6.86 ADI-11815 3.39e57.91e−5 0.23 No binding ADI-11801 7.56e5 4.17e−4 0.55 No bindingADI-10152 2.46e5 9.14e−3 37.2 No binding ADI-10154 1.92e5 8.33e−3 43.4No binding ADI-10155 1.70e5 6.85e−3 40.2 No binding ADI-10157 2.82e67.60e−3 2.69 No binding ADI-10158 4.75e5 1.49e−2 3.14 No bindingADI-10160 6.37e5 2.69e−2 42.2 No binding ADI-10161 5.74e5 1.18e−2 20.6No binding ADI-10163 5.15e5 2.75e−2 53.5 No binding ADI-10164 3.19e51.01e−2 31.5 No binding ADI-10165 5.91e5 1.88e−2 31.8 No bindingADI-10167 1.25e6 3.07e−2 24.4 No binding ADI-10168 3.75e5 1.75e−2 46.6No binding ADI-10173 2.12e5 1.25e−2 58.8 No binding ADI-11802 4.9e5 1.54e−3 3.14 No binding ADI-11812 3.96e5 4.86e−4 1.23 No bindingADI-11825 2.13e6 3.8e−3  1.78 No binding ADI-11826 1.76e6 4.63e−3 2.63No binding ADI-11828 1.32e6 3.51e−3 2.67 1.03e6 5.48e−2 53.5 ADI-118398.74e5 1.62e−2 18.6 No binding Lintuzumab 7.35e5 1.22e−2 16.7 No binding

Mapping of Binding Interface to Individual Domains of CD33

The binding interface between the Fab fragment of each CD33 antibody toCD33 was mapped. FIGS. 5A-5T show binding of Fab fragments of differentCD33 antibodies to individual domains of human CD33 (V domain and Cdomain). No binding to C domain was observed for any antibody tested.ADI-11815 did not bind to either V or C domain, suggesting that itrequires a unique conformational epitope.

Table 13 shows a comparison between kinetics of binding to full ECD ofhuman CD33 and a V domain. ADI-10159, ADI-11176, ADI-11807, ADI-11830,ADI-11835, ADI-11801, ADI-10155, ADI-11802, ADI-11825, ADI-11826,ADI-11828, and ADI-11839 show similar kinetics suggesting that theepitope for these antibodies are located entirely in the V domain.Reduced binding to V domain is observed for ADI-10177, ADI-11809ADI-11819, ADI-10157, ADI-10158, and ADI-10164, suggesting that theseantibodies bind to a conformational epitope, partially located in the Vdomain.

Kinetics of binding to the C domain of human CD33 was also measured withADI-10152, ADI-10154, ADI-10155, ADI-10157, ADI-10158, ADI-10160,ADI-10161, ADI-10163, ADI-10164, ADI-10165, ADI-10167, ADI-10168,ADI-10173, ADI-11802, ADI-11812, ADI-11825, ADI-11826, ADI-11828, andADI-11839. None of these antibodies bound the C domain of human CD33.

TABLE 13 Biacore analysis of FABs binding to recombinant full-length ECDand V domain of human CD33 performed at 37° C. Asterisk indicatesantibodies that bind to a conformational epitope partially located in Vdomain. Human CD33 V domain Human CD33 k_(a) K_(D) k_(a) K_(D) Antibody(1/Ms) k_(d) (1/s) (nM) (1/Ms) k_(d) (1/s) (nM) ADI-10159 9.05e5 5.0e−3 5.56 4.29e5 1.95e−3 4.53 ADI-10177* 1.56e6 8.57e−2 54.8 5.58e5 6.36e−311.4 ADI-11776 4.52e6 1.43e−2 0.33 1.6e6  1.24e−3 0.78 ADI-11807 3.07e68.47e−4 0.28 1.14e6 3.32e−4 0.29 ADI-11809* 2.6e6  7.69e−4 0.3 1.02e61.23e−6 0.0012 ADI-11819 6.75e6 9.16e−4 0.14 2.84e6 5.65e−5 0.02ADI-11830 6.67e6 2.99e−2 4.5 1.99e6 5.47e−3 2.75 ADI-11835 7.6e6 8.68e−3 1.14 2.6e6  1.43e−3 0.53 ADI-11815 No binding 3.39e5 7.91e−50.23 ADI-11801 1.8e6  1.25e−3 0.69 7.56e5 4.17e−4 0.55 ADI-10152 4.88e54.09e−2 100 2.46e5 9.14e−3 37.2 ADI-10154 1.52e6 3.35e−1 220 1.92e58.33e−3 43.4 ADI-10155 5.48e5 4.67e−2 85.2 1.70e5 6.85e−3 40.2ADI-10157* 1.12e5 1.51e−2 134 2.82e6 7.60e−3 2.69 ADI-10158* 1.04e61.33e−1 127 4.75e5 1.49e−2 3.14 ADI-10160 1.59e6 3.27e−1 206 6.37e52.69e−2 42.2 ADI-10161 1.29e6 1.24e−1 95.8 5.74e5 1.18e−2 20.6 ADI-10163No binding 5.15e5 2.75e−2 53.5 ADI-10164 1.29e6 3.7e−1  286 3.19e51.01e−2 31.5 ADI-10165 No binding 5.91e5 1.88e−2 31.8 ADI-10167 Nobinding 1.25e6 3.07e−2 24.4 ADI-10168 No binding 3.75e5 1.75e−2 46.6ADI-10173 No binding 2.12e5 1.25e−2 58.8 ADI-11802 1.34e6 4.35e−3 3.24.9e5  1.54e−3 3.14 ADI-11812 No binding 3.96e5 4.86e−4 1.23 ADI-118254.93e6 1.95e−2 3.95 2.13e6 3.8e−3  1.78 ADI-11826 5.12e6 2.25e−3 4.381.76e6 4.63e−3 2.63 ADI-11828 3.37e6 2.13e−2 6.3 1.32e6 3.51e−3 2.67ADI-11839 9.19e6 4.1e−1  44.6 8.74e5 1.62e−2 18.6

Antibodies Recognize CD33 Independent of its Glycosylation Status.

The ability of anti-CD33 antibodies to recognize glycosylated CD33 wasassayed. Table 14 shows that antibodies recognize V domain independentof its glycosylation status. Human CD33 is heavily glycosylated with 2glycosylation sites located in the V domain. Differences in theglycosylation level of CD33 in different cells have been reported in theliterature. Glycosylation can potentially disturb antibody binding tothe target. In some samples, the V domain was deglycosylated by PNGasebefore testing. De-glycosylation status was confirmed by a shift onSDS-PAGE and MS. All antibodies tested in Table 14 bound todeglycosylated V CD33 similarly to the fully glycosylated version exceptfor ADI-10163, ADI-10165, ADI-10167, and ADI-10173.

TABLE 14 Biacore analysis of FABs binding to fully glycosylated vsdeglycosylated V domain performed at 37° C. Deglycosylated V domain Vdomain K_(D) K_(D) Antibody k_(a) (1/Ms) k_(d) (1/s) (nM) k_(a) (1/Ms)k_(d) (1/s) (nM) ADI-10159 9.78e5 1.2e−2  12.3 9.05e5 5.0e−3  5.56ADI-10177 1.88e6 7.01e−2 38.2 1.56e6 8.57e−2 54.8 ADI-11776 4.85e61.36e−3 0.28 4.52e6 1.43e−2 0.33 ADI-11807 3.9e6  7.45e−4 0.19 3.07e68.47e−4 0.28 ADI-11809 3.4e6  7.11e−4 0.21 2.6e6  7.69e−4 0.3 ADI-118199.15e6 1.03e−3 0.11 6.75e6 9.16e−4 0.14 ADI-11830 6.8e6  2.69e−2 3.956.67e6 2.99e−2 4.5 ADI-11835 7.94e6 7.79e−3 0.98 7.6e6  8.68e−3 1.14ADI-11801 2.1e6  1.15e−3 0.55 1.8e6  1.25e−3 0.69 ADI-10152 1.05e57.7e−2  76.8 4.88e5 4.09e−2 100 ADI-10154 3.13e5 6.55e−2 209 1.52e63.35e−1 220 ADI-10155 1.25e6 1.32e−1 105 5.48e5 4.67e−2 85.2 ADI-101574.11e5 2.61e−2 63.8 1.12e5 1.51e−2 134 ADI-10158 2.63e6 2.31e−1 88.11.04e6 1.33e−1 127 ADI-10160 1.7e6  2.25e−1 133 1.59e6 3.27e−1 206ADI-10161 2.29e6 1.96e−1 85.6 1.29e6 1.24e−1 95.8 ADI-10163 1.68e62.56e−1 152 No binding ADI-10164 8.11e5 1.16e−1 143 1.29e6 3.7e−1  286ADI-10165 1.3e6  2.03e−1 156 No binding ADI-10167 1.66e6 1.28e−1 77.8 Nobinding ADI-10168 No binding No binding ADI-10173 6.74e5 1.31e−1 200 Nobinding ADI-11802 1.57e6 4.17e−3 2.65 1.34e6 4.35e−3 3.2 ADI-11812 Nobinding No binding ADI-11825 5.78e6 1.8e−2  3.11 4.93e6 1.95e−2 3.95ADI-11826 5.37e6 1.98e−2 3.69 5.12e6 2.25e−3 4.38 ADI-11828  3.588e61.89e−2 4.87 3.37e6 2.13e−2 6.3 ADI-11839 1.65e6 1.16e−1 70.3 9.19e64.1e−1  44.6

CD33 Antibodies Bind to the R69G SNP of CD33.

The ability of anti-CD33 antibodies to recognize the R69G mutation inCD33 was assayed. Although several SNPs have been described for CD33,R69G is particularly prominent, occurring in 39-42% of the population.Table 15 shows antibodies binding to human CD33 containing the R69Gmutation.

TABLE 15 Biacore analysis of FABs binding to CD33 R69G. Human CD33 R69GHuman CD33 k_(a) K_(D) k_(a) K_(D) Antibody (1/Ms) k_(d) (1/s) (nM)(1/Ms) k_(d) (1/s) (nM) ADI-10159 4.26e5 2.43e−3 5.68 4.29e5 1.95e−34.53 ADI-10177 2.3e5  2.01e−3 87.4 5.58e5 6.36e−3 11.4 ADI-11776 1.98e64.26e−4 0.22 1.6e6  1.24e−3 0.78 ADI-11807 5.97e5 2.59e−4 0.43 1.14e63.32e−4 0.29 ADI-11809 8.15e5 1.79e−4 0.22 1.02e6 1.23e−6 0.0012ADI-11819 2.78e6 2.44e−4 0.09 2.84e6 5.65e−5 0.02 ADI-11830 2.27e66.63e−3 2.94 1.99e6 5.47e−3 2.75 ADI-11835 3.07e6 2.05e−3 0.67 2.6e6 1.43e−3 0.53 ADI-11815 3.00e5 1.3e−3  4.34 3.39e5 7.91e−5 0.23 ADI-11801No binding 7.56e5 4.17e−4 0.55 ADI-10152 2.88e5 2.18e−2 75.8 2.46e59.14e−3 37.2 ADI-10154 1.53e5 2.83e−2 186 1.92e5 8.33e−3 43.4 ADI-101552.33e5 1.05e−2 45.0 1.70e5 6.85e−3 40.2 ADI-10157 1.56e6 2.87e−2 17.82.82e6 7.60e−3 2.69 ADI-10158 5.30e5 3.31e−2 62.3 4.75e5 1.49e−2 3.14ADI-10160 8.36e5 5.33e−2 63.7 6.37e5 2.69e−2 42.2 ADI-10161 6.29e53.07e−2 48.9 5.74e5 1.18e−2 20.6 ADI-10163 5.59e5 5.9e−2  106 5.15e52.75e−2 53.5 ADI-10164 3.08e5 3.5e−2  113 3.19e5 1.01e−2 31.5 ADI-101656.9e5  5.02e−2 72.7 5.91e5 1.88e−2 31.8 ADI-10167 1.22e6 8.74e−2 71.61.25e6 3.07e−2 24.4 ADI-10168 4.85e5 3.07e−2 63.5 3.75e5 1.75e−2 46.6ADI-10173 No binding 2.12e5 1.25e−2 58.8 ADI-11802 No binding 4.9e5 1.54e−3 3.14 ADI-11812 3.38e5 2.28e−3 6.74 3.96e5 4.86e−4 1.23 ADI-118251.86e6 6.95e−3 3.74 2.13e6 3.8e−3  1.78 ADI-11826 1.89e6 7.23e−3 3.831.76e6 4.63e−3 2.63 ADI-11828 1.16e6 7.00e−3 6.03 1.32e6 3.51e−3 2.67ADI-11839 No binding 8.74e5 1.62e−2 18.6

CD33 Antibodies Bind to the S128N SNP of CD33

The ability of anti-CD33 antibodies to recognize the S128N mutation inCD33 was assayed. Table 16 shows antibodies binding to human CD33containing the S128N mutation. The binding affinity of ADI-10152,ADI-10154, ADI-10157, ADI-10158, ADI-10163, ADI-10164, ADI-10165,ADI-10167, ADI-10168, and ADI-10173 to human CD33 was impaired by theS128N SNP.

TABLE 16 Biacore analysis of FABs binding to CD33 S128N. Human CD33S128N Human CD33 Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (nM) k_(a)(1/s) k_(d) (1/s) K_(D) (nM) ADI-10152 2.74e5 3.39e−2 124 2.46e5 9.14e−337.2 ADI-10154 1.93e5 5.39e−2 279 1.92e5 8.33e−3 43.4 ADI-10155 1.87e51.59e−2 85 1.70e5 6.85e−3 40.2 ADI-10157 5.65e4 9.24e−3 163 2.82e67.60e−3 2.69 ADI-10158 5.73e5 4.17e−2 72.8 4.75e5 1.49e−2 3.14 ADI-101607.86e5 6.73e−2 85.7 6.37e5 2.69e−2 42.2 ADI-10161 6.05e5 3.85e−2 63.65.74e5 1.18e−2 20.6 ADI-10163 5.07e5 7.48e−2 147 5.15e5 2.75e−2 53.5ADI-10164 1.93e5 5.39e−2 279 3.19e5 1.01e−2 31.5 ADI-10165 6.41e55.82e−2 90.7 5.91e5 1.88e−2 31.8 ADI-10167 1.28e6 1.27e−1 98.6 1.25e63.07e−2 24.4 ADI-10168 4.04e5 4.69e−2 116 3.75e5 1.75e−2 46.6 ADI-101731.83e5 3.42e−2 187 2.12e5 1.25e−2 58.8 ADI-11802 6.62e5 5.90e−4 0.8094.9e5  1.54e−3 3.14 ADI-11812 5.56e5 1.12e−3 2.01 3.96e5 4.86e−4 1.23ADI-11825 2.10e6 6.49e−3 3.09 2.13e6 3.8e−3  1.78 ADI-11826 2.17e65.74e−3 2.65 1.76e6 4.63e−3 2.63 ADI-11828 1.37e6 7.42e−3 5.41 1.32e63.51e−3 2.67 ADI-11839 9.63e5 2.84e−2 29.5 8.74e5 1.62e−2 18.6

ADI-11815 Recognizes a Unique Conformational Epitope.

The binding epitope of the CD33-binding domain of ADI-11815 was assayed.FIG. 6 and Table 17 demonstrate that ADI-11815 has a uniqueconformational epitope. This antibody binds to the full-length ECD ofhuman CD33, but not to individual domains and does not cross-block withlintuzumab.

TABLE 17 Kinetic parameters of ADI-11815 Fab binding to differentdomains and SNP R69G of human CD33. hCD33 V domain C domain hCD33 R69GSNP Cross- k_(a) k_(d) K_(D) k_(a) k_(d) K_(D) k_(a) k_(d) K_(D) k_(a)k_(d) K_(D) blocking with (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) (1/Ms)(1/s) (nM) (1/Ms) (1/s) (nM) lintuzumab ADI- 3.39e5 7.91e−5 0.23 Nobinding No binding 3.00e5 1.3e−3 4.34 No 11815ADI-11801 Binds to a Unique Epitope that Includes R69.

The binding epitope of CD33-binding domain including ADI-11815 wasassayed. FIG. 7 and Table 18 demonstrate the epitope on CD33 thatADI-11801 recognize. Its binding to the human CD33 ECD is abrogated byR69G mutation.

TABLE 18 Kinetics of ADI-11801 Fab binding to different domains of CD33and SNP R69G. Cross- hCD33 V domain C domain hCD33 R69G SNP blockingk_(a) k_(d) K_(D) k_(a) k_(d) K_(D) k_(a) k_(d) K_(D) k_(a) k_(d) K_(D)with (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) (1/Ms) (1/s)(nM) lintuzumab AD-11801 7.56e5 4.17e−4 0.55 1.80e6 1.25e−3 0.69 Nobinding No binding No

Example 3: Assessment of TriNKET Binding to Cell Expressing Human NKG2D

The ability of TriNKETs that include an NKG2D-binding domain and aCD33-binding domain to bind to NKG2D was assayed. EL4 cells transducedwith human NKG2D and human KHYG-1 cells were used to test binding tocell-expressed human NKG2D. TriNKETs were diluted to the topconcentration, and then diluted serially. The mAb or TriNKET dilutionswere used to stain cells, and binding of the TriNKET or mAb was detectedusing a fluorophore-conjugated anti-human IgG secondary antibody. Cellswere analyzed by flow cytometry, binding MFI was normalized to secondaryantibody controls to obtain fold over background values.

FIGS. 10A-10B show binding of CD33-targeting TriNKETs to human NKG2Dexpressed on EL4 (top) or KHYG-1 (bottom) cells. FOB binding signal wassimilar on both EL4-hNKG2D cells and KHYG-1 cells, the ranking ofbinding between clones was also maintain on the two cell lines. FIG. 10Ashows binding of CD33-targeting TriNKETs to human NKG2D expressed on EL4cells. FIG. 10B shows binding of CD33-targeting TriNKETs to human NKG2Dexpressed on KHYG-1 cells. FOB binding signal was similar on bothEL4-hNKG2D cells and KHYG-1 cells, the ranking of binding between cloneswas also maintain on the two cell lines.

Example 4: Assessment of TriNKET or mAb Binding to Cell Expressed HumanCancer Antigens

The ability of TriNKETs that include an NKG2D-binding domain and aCD33-binding domain to bind to CD33 was assayed. The Molm-13, human AMLcell line, was used to assess binding of monoclonal antibodies to CD33expressed on the cell surface. mAbs were diluted to 2 μg/mL, and mAbdilutions were used to stain cells. Bound antibody was detected using afluorophore conjugated anti-human IgG secondary antibody. Cells wereanalyzed by flow cytometry, binding to cell-expressed CD33 was comparedto isotype stained and unstained cell populations.

Human cancer cell lines expressing CD33 were used to assess tumorantigen binding of TriNKETs derived from different NKG2D-targetingclones. The human AML cell line Molm-13 was used to assess binding ofTriNKETs to cell expressed CD33. TriNKETs were diluted, and wereincubated with the respective cells. Binding of the TriNKET was detectedusing a fluorophore conjugated anti-human IgG secondary antibody. Cellswere analyzed by flow cytometry, binding MFI to cell expressed CD33 wasnormalized to secondary antibody controls to obtain fold over backgroundvalues.

The ability of CD33 TriNKETs to bind CD33 expressed on Molm-13 cells wastested. FIG. 8 shows binding of TriNKETs targeting CD33 to Molm-showsbinding MFI of six anti-CD33 antibodies to CD33 expressed on Molm-13cells. All six antibodies bound to cell-expressed CD33. Five of the sixantibodies show higher MFI binding signal compared to Lintuzumab.

The ability of CD33 TriNKETs to induce rested NK cell-mediated killingof Molm-13 AML cells was tested. Four different CD33-targeting domainswere used with five NKG2D-targeting domains to make a total of 20different TriNKETs. Regardless of the NKG2D-targeting domain used in theTriNKET, binding to CD33 was conserved for a single CD33-targetingdomain.

Example 5: Assessment of TriNKET or mAb Internalization

Internalization of TriNKETs after binding to CD33 on cell surface wasassayed. The Molm-13, human AML cell line, was used to assessinternalization of monoclonal antibodies bound to CD33 expressed on thecell surface. Monoclonal antibodies were diluted to 2 μg/mL, and mAbdilutions were used to stain cells. Following surface staining of CD33samples were split, half the sample was placed at 37° C. overnight tofacilitate internalization, with the other half of the sample boundantibody was detected using a fluorophore conjugated anti-human IgGsecondary antibody. Cells were fixed after staining with the secondaryantibody, and were stored at 4° C. overnight for analysis on thefollowing day. After 24 hours at 37° C. samples were removed from theincubator, and bound antibody on the surface of the cells was detectedusing a fluorophore conjugated anti-human IgG secondary antibody.Samples were fixed and all samples were analyzed on the same day.Internalization of antibodies was calculated as follows: %internalization=(1−(sample MFI 24 hours/baseline MFI))*100%.

FIG. 9 shows internalization of anti-CD33 antibodies bound to thesurface of Molm-13 cells after 24 hours. All the anti-CD33 antibodiesshowed similar internalization after 24 hours. Lintuzumab showedslightly higher internalization compare to other anti-CD33 antibodies.

Example 6: Activation of Primary NK Cells by TriNKETs

The ability of TriNKETs that include an NKG2D-binding domain and aCD33-binding domain to activate primary NK cells was assayed. PBMCs wereisolated from human peripheral blood buffy coats using density gradientcentrifugation. Isolated PBMCs were washed and prepared for NK cellisolation. NK cells were isolated using a negative selection techniquewith magnetic beads, purity of isolated NK cells was typically >90%CD3-CD56+. Isolated NK cells were cultured in media containing 100 ng/mLIL-2 for activation or rested overnight without cytokine. IL-2-activatedNK cells were used either 24-48 hours later; rested NK cells were alwaysused the day after purification.

Human cancer cell lines expressing a cancer target of interest wereharvested from culture, and cells were adjusted to 2×10⁶/mL. Monoclonalantibodies or TriNKETs targeting the cancer target of interest werediluted in culture media. Rested and/or activated NK cells wereharvested from culture, cells were washed, and were resuspended at2×10⁶/mL in culture media. IL-2, and fluorophore-conjugated anti-CD107awas added to the NK cells for the activation culture. Brefeldin-A andmonensin were diluted into culture media to block protein transport outof the cell for intracellular cytokine staining. Into a 96-well plate 50μl of tumor targets, mAbs/TriNKETs, BFA/monensin, and NK cells wereadded for a total culture volume of 200 μl. Plate was cultured for 4hours before samples were prepared for FACS analysis.

Following the 4 hour activation culture, cells were prepared foranalysis by flow cytometry using fluorophore-conjugated antibodiesagainst CD3, CD56 and IFNγ. CD107a and IFNγ staining was analyzed inCD3-CD56+ populations to assess NK cell activation.

FIG. 12 shows TriNKET-mediated activation of rested human NK cells inco-culture with CD33-expressing THP-1 AML cells. Human NK cellactivation was assessed using IFNγ production and CD107a degranulationas markers for activation. All TriNKETs and monoclonal antibodiesactivated human NK cells above the isotype control. Similar activity wasobserved for four different anti-CD33 antibodies. TriNKET activity wasdependent upon the NKG2D-targeting clone, some clones provided betterTriNKET mediated activation than others. NKG2D clones provided similaractivity with each of the anti-CD33 targeting domains.

Example 7: Primary Human NK Cell Cytotoxicity Assay

The ability of TriNKETs that include an NKG2D-binding domain and aCD33-binding domain to induce cytotoxicity of NK cells againstCD33-expressing cells was assayed. PBMCs were isolated from humanperipheral blood buffy coats using density gradient centrifugation.Isolated PBMCs were washed and prepared for NK cell isolation. NK cellswere isolated using a negative selection technique with magnetic beads,purity of isolated NK cells was typically >90% CD3-CD56+. Isolated NKcells were cultured in media containing 100 ng/mL IL-2 or were restedovernight without cytokine. IL-2-activated or rested NK cells were usedthe following day in cytotoxicity assays.

KHYG-1 cells were maintained in 10% HI-FBS-RPMI-1640 with 10 ng/mL IL-2.The day before use as effector cells in killing assays KHYG-1 cells wereharvest from culture, and cells were washed out of the IL-2-containingmedia. After washing KHYG-1 cells were resuspended in 10%HI-FBS-RPMI-1640, and were rested overnight without cytokine.

DELFIA Cytotoxicity Assay:

Human cancer cell lines expressing a target of interest were harvestedfrom culture, cells were washed with HBS, and were resuspended in growthmedia at 10⁶/mL for labeling with BATDA reagent (Perkin Elmer AD0116).Manufacturer instructions were followed for labeling of the targetcells. After labeling cells were washed 3× with HBS, and wereresuspended at 0.5-1.0×10⁵/mL in culture media. To prepare thebackground wells an aliquot of the labeled cells was put aside, and thecells were spun out of the media. 100 μl of the media was carefullyadded to wells in triplicate to avoid disturbing the pelleted cells. 100μl of BATDA labeled cells were added to each well of the 96-well plate.Wells were saved for spontaneous release from target cells, and wellswere prepared for max lysis of target cells by addition of 1% Triton-X.Monoclonal antibodies or TriNKETs against the tumor target of interestwere diluted in culture media and 50 μl of diluted mAb or TriNKET wasadded to each well. Rested and/or activated NK cells were harvested fromculture, cells were washed, and were resuspended at 10⁵-2.0×10⁶/mL inculture media depending on the desired E:T ratio. 50 μl of NK cells wasadded to each well of the plate to make a total of 200 μl culturevolume. The plate was incubated at 37° C. with 5% CO2 for 2-3 hoursbefore developing the assay.

After culturing for 2-3 hours, the plate was removed from the incubatorand the cells were pelleted by centrifugation at 200 g for 5 minutes. 20μl of culture supernatant was transferred to a clean microplate providedfrom the manufacturer and 200 μl of room temperature europium solutionwas added to each well. The plate was protected from the light andincubated on a plate shaker at 250 rpm for 15 minutes. Plate was readusing either Victor 3 or SpectraMax i3X instruments. % Specific lysiswas calculated as follows: % Specific lysis=((Experimentalrelease−Spontaneous release)/(Maximum release−Spontaneousrelease))*100%.

FIG. 13 and FIG. 14 show human NK cell killing of Molm-13 (FIG. 13 ) andTHP-1 (FIG. 14 ) AML target cells mediated by CD33-targeting TriNKETs.Human NK cell killing of Molm-13 AML target cells mediated byCD33-targeting TriNKETs was assayed (FIG. 13 ). Rested NK effector cellswere used with Molm-13 target cells. Activated human NK effector cellsgave higher background killing, compared to rested human NK effectorcells. With NK effector cells, TriNKETs were able to increase lysisagainst Molm-13 AML target cells. Similar activity of each TriNKET wasobserved with rested and activated human NK cells, as well as withMolm-13 target cells.

Human NK cell killing of THP-1 AML target cells, mediated byCD33-targeting TriNKETs was assayed. Activated human NK effector cellswere used with THP-1 cells (FIG. 14 ), which resulted in higherbackground killing, compared to rested human NK effector cells. With NKeffector cells, TriNKETs were able to increase lysis against THP-1 AMLtarget cells. Similar activity of each TriNKET was observed with restedand activated human NK cells, as well as with THP-1 target cells.

Thus, with NK effector cells TriNKETs were able to increase lysisagainst Molm-13 (FIG. 13 ) and THP-1 (FIG. 14 ) AML target cells.Similar activity of each TriNKET was observed with rested and activatedhuman NK cells, as well as with Molm-13 (FIG. 13 ) and THP-1 (FIG. 14 )target cells.

FIG. 15A, FIG. 16 , and FIG. 17A show KHYG-1 effector cell killing ofMolm-13 (FIG. 15A), EOL-1 (FIG. 16 ), and THP-1 (FIG. 17A) human AMLtarget cells respectively. KHYG-1 cells were demonstrated to expresssurface NKG2D, but do not express CD16. Therefore, TriNKET mediatedkilling here is dependent upon NKG2D mediated activation of the KHYG-1effector cells. TriNKETs were able to mediate KHYG-1 effector cellkilling of all three human AML target cell lines. Similar, TriNKETactivity was demonstrated on all three target cell lines.

TriNKET mediated cytotoxicity of rested human NK cells was also tested.FIG. 15B and FIG. 17B show that TriNKETs also mediated cytotoxicity ofrested human NK cells against Molm-13 (FIG. 15B) and THP-1 (FIG. 17B)human AML cells. FIG. 15B shows that TriNKETs mediated rested human NKcell killing of Molm-13 human AML cells. In FIG. 15B the rested human NKeffector cell (E) to target cancer cell (T) ratio (E:T) was 10:1. TheE:T ratio may reflect differences in the maximal % lysis.

TriNKETs also mediated rested human NK cell killing of THP-1 targetcells, which express the high-affinity FcγRI. FIG. 17B shows thatTriNKETs mediated rested human NK cell killing of THP-1 human AML cells,in which the E:T was 5:1.

Example 8: Assessment of TriNKET Binding to Cells Expressing Human NKG2D

I07 mAb was identified as a monoclonal antibody with high bindingaffinity to CD33. The heavy and light chain amino acid sequences ofI07-F405L, an Fc variant of I07, are provided below. The I07-F405L mAbincluded a substitution of Leu for Phe at position 405 (under EUnumbering) in the Fc CH3 domain. A Lys may be optionally included at theC-terminus of the heavy chain.

I07-F405L mAb heavy chain [SEQ ID NO: 199]EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKGLEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGI07-F405L mAb light chain [SEQ ID NO: 200]DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSQSYPPITFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

A49-F3′-TriNKET-I07, a TriNKET derivative of I07 mAb, is described inSection II—Multi-specific Binding Proteins. The amino acid sequences ofthis TriNKET are provided in SEQ ID NOs: 187, 189, and 190.

The ability of A49-F3′-TriNKET-I07 to bind to NKG2D was assessed usingEL4 cells transduced with human NKG2D (EL4-hNKG2D). Briefly,A49-F3′-TriNKET-I07 and I07-F405L mAb were serially diluted. EL4-hNKG2Dcells were incubated with the diluted TriNKET or mAb solutions. Bindingof the TriNKET or mAb to the EL4 cells was detected using afluorophore-conjugated anti-human IgG secondary antibody. The cells wereanalyzed by flow cytometry, and fold over background values werecalculated by normalizing binding MFI to the MFI of a control group inwhich the cells were incubated with the secondary antibody only.

As shown in FIG. 35 , I07-F405L mAb showed no binding to EL4-hNKG2Dcells. A49-F3′-TriNKET-I07 demonstrated weak binding to EL4-hNKG2Dcells, without reaching saturation even at the high concentrations of100 μg/mL.

Example 9: Assessment of TriNKET Binding to Cells Expressing Human CD33

The ability of A49-F3′-TriNKET-I07 to bind to CD33 was assessed usinghuman cancer cell lines expressing CD33. Briefly, human AML cell linesMv4-11 and Molm-13, which expressed CD33, were incubated with seriallydiluted A49-F3′-TriNKET-I07 and I07-F405L mAb solutions. Binding ofA49-F3′-TriNKET-I07 or I07-F405L mAb to the AML cells was detected usinga fluorophore-conjugated anti-human IgG secondary antibody. The cellswere analyzed by flow cytometry, and fold over background values werecalculated by normalizing binding MFI to the MFI of a control group inwhich the cells were incubated with the secondary antibody only.

As shown in FIGS. 36A and 36B, A49-F3′-TriNKET-I07 exhibited a decreasein binding potency by three to four fold compared to I07-F405L mAb onboth Molm-13 and Mv4-11 cells. A49-F3′-TriNKET-I07 was also found tobind to the cells with a higher maximum fold over background compared toI07-F405L mAb.

Example 10: Assessment of TriNKET Internalization

Internalization of A49-F3′-TriNKET-I07 and I07-F405L mAb upon binding toCD33 was assessed using human AML cell lines EOL-1 and Molm-13, whichexpressed CD33 on the cell surface. Briefly, the cells were incubatedwith 2 μg/mL A49-F3′-TriNKET-I07 or I07-F405L mAb for 20 minutes at roomtemperature. The cell samples were then split into three portions. Thefirst and second portions were placed at 37° C. for 2 hours and 24hours, respectively, to allow antibody internalization. Then the cellswere incubated with a fluorophore-conjugated anti-human IgG secondaryantibody, and were fixed for flow cytometry analysis. The third portionof the cell samples, used to set the baseline level, was incubated withthe fluorophore-conjugated anti-human IgG secondary antibody withoutincubation at 37° C. The cells were fixed after staining with thesecondary antibody, and were stored at 4° C. for analysis on thefollowing day (when the first half of the samples were ready). Theamount of A49-F3′-TriNKET-I07 and I07-F405L mAb bound to the cellsurface was analyzed by flow cytometry on the same day. Internalizationof antibodies was calculated as: % internalization=(1-(MFI of 24-hoursample/MFI of baseline sample))×100%.

As shown in FIGS. 37A and 37B, internalization of A49-F3′-TriNKET-I07and I07-F405L mAb after engagement of CD33 increased overtime on EOL-1and Molm-13 cells. In both cells, I07-F405L mAb was internalized morerapidly and to a greater extent than A49-F3′-TriNKET-I07.

Example 11: Activation of Primary NK Cells by TriNKET

The ability of A49-F3′-TriNKET-I07 to elicit cytotoxicity of primary NKcells against human AML cells was assessed using the DELFIA cytotoxicityassay. Briefly, PBMCs were isolated from human peripheral blood buffycoats using density gradient centrifugation. The isolated PBMCs werewashed, and NK cells were isolated using a negative selection techniquewith magnetic beads. The purity of the isolated NK cells wastypically >90% CD3⁻CD56⁺. The isolated NK cells were rested withoutcytokine overnight.

On the following day, human AML cell lines Molm-13, THP-1, and EOL-1were harvested from culture. The AML cells were washed with HBS, andwere resuspended in growth media at 10⁶ cells/mL for labeling with BATDAreagent (Perkin Elmer AD0116) following the manufacturer instructions.After labeling, the AML cells were washed three times with HBS, and wereresuspended at 0.5-1.0×10⁵ cells/mL in culture media. 100 μl of BATDAlabeled cells were added to each well of a 96-well plate.

The tested TriNKET or mAb was diluted in culture media, and 50 μl ofdiluted TriNKET or mAb was added to each well. Rested NK cells wereharvested from culture, washed, and resuspended at 10⁵-2.0×10⁶ cells/mLin culture media to attain a desired E:T ratio of 5:1. 50 μl of NK cellswere added to each well of the plate to make a total of 200 μl culturevolume in each well. The plate was incubated at 37 C with 5% CO₂ for 2-3hours.

After the culturing, the plate was removed from the incubator, and thecells were pelleted by centrifugation at 200×g for 5 minutes. 20 μl ofculture supernatant was transferred to a clean microplate provided fromthe manufacturer. Supernatant from the labeled cells incubated alone wasused to measure spontaneous release of TDA. Supernatant from labeledcells incubated with 1% Triton-X was used to measure maximum lysis ofthe target cells. Supernatant from the labeled cells prior to the 2-3hours of incubation was used to measure the background and for qualitycontrol purposes.

200 μl of room temperature europium solution was added to each wellcontaining culture supernatant. The plate was protected from light andincubated on a plate shaker at 250 rpm for 15 minutes. Fluorescence wasmeasured using a Victor 3 or SpectraMax i3X instrument.

The fluorescent levels represented lysis of the target cells. The valuesof % specific lysis were calculated as: % specific lysis=((Experimentalrelease−Spontaneous release)/(Maximum release−Spontaneousrelease))×100%.

A49-F3′-TriNKET-I07 and several monoclonal antibodies were tested inthis assay. The monoclonal antibodies include I07-F405L mAb, I07-DE mAb,lintuzumab-GA, and 280-31-01(mut)-DE. I07-DE mAb is a variant of I07mAb, with S239D and I332E substitutions in the Fc to enhance ADCCactivity (bold-underlined in the sequence below). The amino acidsequence of I07-DE heavy chain is shown below, optionally with a Lys atthe C-terminus.

I07-DE mAh heavy chain [SEQ ID NO: 201]EVQLVESGGGLVQPGGSLRLSCAASGFTFGSYWMSWVRQAPGKGLEWVATIKQDGSEKSYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPLNAGELDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP D VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP E EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

The light chain of I07-DE mAb is identical to that of I07-F405L mAb [SEQID NO:200].

280-31-01(mut)-DE is a variant of antibody clone 280-31-01 (mut)disclosed in WO2012045752, with S239D and I332E substitutions in the Fcto enhance ADCC activity (bold-underlined in the sequence below). Theamino acid sequences of 280-31-01(mut)-DE heavy chain and light chainare shown below, optionally with a Lys at the C-terminus of the heavychain.

280-31-01(mut)-DE heavy chain (SEQ ID NO: 202)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYAISWVRQAPGQGLEWMGRIIPILGVADYAQKFQGRVTITADKSTRTAYMELSSLRSEDTAVYYCARNWADAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP D VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP E EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG280-31-01(mut)-DE light chain (SEQ ID NO: 203)DIQLTQSPSSLSASVGDRVTITCRASQGISSVLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFDSSITFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC

An SNP in the CD16 gene can result in V158 or F158 variants of the humanCD16 protein. CD16 with F158 is known to have reduced binding affinityof CD16 to Fc than CD16 with V158, thereby decreasing antibody-dependentcell-mediated cytotoxicity (ADCC). NK cells having CD16-F158 aretherefore less responsive to CD16 stimulation than NK cells expressingCD16-V158. Indeed, as shown in FIG. 38A, I07-F405L mAb only led to a lowlevel of killing of Molm-13 cells by NK cells that only expressed thelow affinity CD16 variant (CD16^(F/F)). In comparison,A49-F3′-TriNKET-I07 mediated more potent NK cell killing with a higherspecific lysis of Molm-13 cells, likely due to its additional ability toengage NK cells by NKG2D binding. Similarly, when incubated with NKcells that had this SNP in one allele (CD16^(V/F)), A49-F3′-TriNKET-I07exhibited stronger activity in killing EOL-1 cells than I07-F405L mAb(FIG. 38B).

THP-1 cells express FcγRI, which can bind to IgG1 Fc at high affinity.The competition for Fc binding by target cells can further reduce NKcell killing. Therefore, as shown in FIGS. 38C and 38D, when THP-1 cellswere incubated with CD16^(F/F) NK cells in the presence of I07-F405L orlintuzumab-GA mAb, no specific lysis of THP-1 cells was observed. EvenI07-DE, an ADCC-enhancing variant of I07, failed to elicit NK cellcytotoxicity. Among the monoclonal antibodies tested, only280-31-01(mut)-DE, an ADCC-enhancing variant of antibody clone 280-31-01(mut) disclosed in WO2012045752, exhibited cell killing activity at highconcentrations. Remarkably, A49-F3′-TriNKET-I07 mediated more potent NKcell killing with a higher specific lysis of THP-1 cells, likely due toits additional ability to engage NK cells by NKG2D binding.

Example 12: Activation of Primary CD8⁺ T Cells by TriNKET

NKG2D is expressed on NK cells and many T cells, including CD8⁺ T cells.The ability of A49-F3′-TriNKET-I07 to elicit cytotoxicity of primaryCD8⁺ T cells against human AML cells was assessed using the DELFIAcytotoxicity assay.

Briefly, human peripheral blood mononuclear cells (PBMCs) were isolatedfrom human peripheral blood buffy coats using density gradientcentrifugation. The isolated PBMCs were stimulated with 1 μg/mLConcanavalin A (ConA) at 37° C. for 18 hours. Then ConA was removed, andthe PBMCs were cultured with 25 unit/mL IL-2 at 37° C. for 4 days. CD8⁺T cells were purified using a negative selection technique with magneticbeads, then cultured in media containing 10 ng/mL IL-15 at 37° C. for7-13 days.

The primary human effector CD8⁺ T cells generated above werecharacterized for cell markers. The cells were stained withfluorophore-conjugated antibodies against CD3, CD8, NKG2D, and CD16, andanalyzed by flow cytometry. As shown in FIG. 39 , the isolated CD8⁺ Tcells had high purity, as more than 99% of them were positive of CD3,CD8, and NKG2D expression, and were negative of CD16 expression.

To assess the ability of A49-F3′-TriNKET-I07 to elicit cytotoxicity ofprimary CD8⁺ T cells, Molm-13 cells were harvested from culture, washed,and resuspended in growth media at 10⁶ cells/mL. The cells were labeledwith BATDA reagent (Perkin Elmer AD0116) following manufacturerinstructions. After labeling, the cells were washed three times withHBS, and were resuspended at 0.5×10⁵ cells/mL in culture media. 100 μlof BATDA labeled cells were added to each well of a 96-well plate. 50 μlof serially diluted monoclonal antibody or TriNKET was added to eachwell.

CD8⁺ effector T cells were harvested from culture, washed, andresuspended at 5×10⁶ cells/mL in culture media. 50 μl of CD8⁺ T cellswere added to each well of the plate to reach an E:T ratio of 50:1 and atotal of culture volume of 200 μl. The plate was incubated at 37° C.with 5% CO₂ for 3.5 hours. After incubation, the cells were pelleted bycentrifugation at 500×g for 5 minutes. 20 μl of culture supernatant wastransferred to a clean microplate provided from the manufacturer.Supernatant from the labeled cells incubated alone was used to measurespontaneous release of TDA. Supernatant from labeled cells incubatedwith 1% Triton-X was used to measure maximum lysis of the target cells.

200 μl of room temperature europium solution was added to each well. Theplate was protected from light and incubated on a plate shaker at 250rpm for 15 minutes. Fluorescence was measured using a SpectraMax i3Xinstrument.

The fluorescent levels represented lysis of the target cells. The valuesof % specific lysis were calculated as: % specific lysis=((Experimentalrelease−Spontaneous release)/(Maximum release−Spontaneousrelease))×100%.

As shown in FIGS. 40A and 40B, A49-F3′-TriNKET-I07 enhanced thecytotoxic activity of human primary CD8⁺ T cells in a dose-dependentmanner. A49-F3′-TriNKET-H76, a protein described in Section II—Multi-specific Binding Proteins (having polypeptides comprising thesequences of SEQ ID NOs: 197, 189, and 190), was also active under theconditions but exhibited less potency than A49-F3′-TriNKET-I07.Monoclonal antibody I07-F405L and a non-target TriNKET did not show thisactivity.

Example 13: Binding of TriNKET to Monocytes

The expression of CD33 on blood cells is assessed by flow cytometryusing the method described in Example 9. Briefly, human whole blood wasincubated with A49-F3′-TriNKET-I07 or human IgG1 isotype controlantibody conjugated to a fluorophore. Binding of A49-F3′-TriNKET-I07 orthe isotype control antibody was detected by flow cytometry. To assessthe binding levels on specific types of cells, fluorophore-conjugatedantibodies that bind to surface markers of NK cells, CD8⁺ T cells, CD4⁺T cells, B cells, and monocytes were added to the incubation, and thepresence or absence of binding of these antibodies were used for gatingwhen analyzing the flow cytometry data.

As shown in FIGS. 41A-41E, the binding of A49-F3′-TriNKET-I07 to NKcells was weak compared to a non-target human IgG1 isotype antibodycontrol, whereas strong binding of the TriNKET to CD33⁺ monocytes wasobserved.

Example 14: Long-Term NK Cell Cytotoxicity Mediated by TriNKET

NK cells have a natural ability to sense transformed or stressed cellsand kill them, but do not kill healthy cells. We tested the ability ofA49-F3′-TriNKET-I07 to preserve the natural selective NK cellcytotoxicity using Molm-13 AML cells and human primary monocytes astarget cells. The Molm-13 cells were obtained from DSMZ cell bank. Thehuman primary monocytes were isolated from human peripheral blood.Briefly, PBMCs were isolated from human peripheral blood buffy coatsusing density gradient centrifugation. Monocytes were isolated bynegative selection. CD33 expression on human primary monocytes andMolm-13 AML cells were confirmed by flow cytometry analysis (FIGS.42A-42B).

Human primary NK cells were isolated from human peripheral blood.Briefly, PBMCs were isolated from human peripheral blood buffy coatsusing density gradient centrifugation. NK cells were isolated bynegative selection. To distinguish target cells from NK cells in theco-culture, the target cells were fluorescently labeled. Specifically,the isolated monocytes were labeled with IncuCyte CytoLight Rapidlive-cell labeling reagent according manufacturer's recommendations. TheMolm-13 cells were infected with lentivirus encoding nuclear GFP, andcells with stable expression were selected with puromycin.

Isolated NK cells and target cells were mixed at an E:T ratio of 10:1 inthe presence of 20 nM A49-F3′-TriNKET-I07. Non-specific activation of NKcells in the co-cultures was conducted in parallel as a positive controlgroup for AML cell killing. The mixture was added to an Ibidi μ-slide.Time-lapse images for the phase and green channels were collected everyhour, with 3 images per sample, using an IncuCyte S3 instrument. Theimages were analyzed using the IncuCyte S3 software. Live target cellswere detected by green fluorescence, and the number of green cells ateach time point was normalized to the number of green cells at time 0from the same sample.

As shown in FIGS. 43A and 43B, Molm-13 AML cells were able toproliferation in the presence of the NK cells alone, but target celloutgrowth was substantially inhibited by A49-F3′-TriNKET-I07. Bycontrast, A49-F3′-TriNKET-I07 did not mediate human NK cell killing ofnormal monocytes in the long-term co-culture. The activity ofA49-F3′-TriNKET-I07 was similar to that of PMA+ionomycin, which alsopreserves the natural selectivity of NK cells. These results suggestthat A49-F3′-TriNKET-I07 selectively compromised cancer cells, andpotentially had a wide therapeutic window.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andthe range of equivalency of the claims are intended to be embracedtherein.

1-46. (canceled)
 47. A protein comprising: (a) a first antigen-binding site comprising a Fab fragment that binds NKG2D, wherein the Fab fragment comprises the amino acid sequence of SEQ ID NO:114, the amino acid sequence of SEQ ID NO:115, the amino acid sequence of SEQ ID NO:116, the amino acid sequence of SEQ ID NO:111, the amino acid sequence of SEQ ID NO:112, and the amino acid sequences of SEQ ID NO:113; (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33, wherein the scFv comprises the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:49, the amino acid sequence of SEQ ID NO:50, the amino acid sequence of SEQ ID NO:45, the amino acid sequence of SEQ ID NO:46, and the amino acid sequences of SEQ ID NO:47; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16.
 48. The protein according to claim 47, wherein the C-terminus of the Fab fragment is linked to the antibody Fc domain or a portion thereof sufficient to bind CD16.
 49. The protein according to claim 47, wherein the first antigen-binding site that binds NKG2D comprises a heavy chain variable domain amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:87 and a light chain variable domain amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:88.
 50. The protein according to claim 47, wherein the light chain variable domain of the scFv of the second antigen-binding site is positioned at the N-terminus of the heavy chain variable domain of the scFv.
 51. The protein according to claim 50, wherein the light chain variable domain of the scFv of the second antigen-binding site is linked to the heavy chain variable domain of the scFv via a linker, and wherein the linker amino acid sequence comprises the amino acid sequence of (GlyGlyGlyGlySer)₄ ((G4S)₄) (SEQ ID NO:186).
 52. The protein according to claim 47, wherein a disulfide bridge is formed between the heavy chain variable domain of the scFv and the light chain variable domain of the scFv.
 53. The protein according to claim 47, wherein the C-terminus of the heavy chain variable domain of the scFv of the second antigen-binding site is linked to the N-terminus of the antibody Fc domain or a portion thereof.
 54. The protein according to claim 53, wherein the C-terminus of the heavy chain variable domain of the scFv of the second antigen-binding site is linked to the N-terminus of the antibody Fc domain or a portion thereof via a hinge comprising Ala-Ser or Gly-Ala-Ser.
 55. The protein according to claim 47, wherein the scFv of the second antigen-binding site comprises a heavy chain variable domain amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:9 and a light chain variable domain amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:10.
 56. The protein according to claim 47, wherein the scFv of the second antigen-binding site comprises the amino acid sequence of SEQ ID NO:188.
 57. The protein according to claim 47, wherein the antibody Fc domain sufficient to bind CD16 comprises a hinge region and a CH2 domain of a human IgG1 antibody.
 58. The protein according to claim 47, wherein the antibody Fc domain sufficient to bind CD16 comprises an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody.
 59. The protein according to claim 47, wherein the antibody Fc domain sufficient to bind CD16 comprises an amino acid sequence at least 90% identical to the Fc domain of a human IgG1 and differs from the Fc domain of a human IgG1 at one or more positions that is Q347, Y349, T350, L351, 5354, E356, E357, K360, Q362, 5364, T366, L368, K370, N390, K392, T394, D399, 5400, D401, F405, Y407, K409, T411, or K439, numbered according to the Kabat numbering.
 60. The protein according to claim 47, wherein the antibody Fc domain sufficient to bind CD16 is an Fc domain of a human IgG1 comprising substitutions for forming a heterodimer.
 61. The protein according to claim 47, wherein the antibody Fc domain sufficient to bind CD16 is an Fc domain of a human IgG1 comprising Y349C, K360E and K409W substitutions, numbered according to the Kabat numbering.
 62. The protein according to claim 47, wherein the antibody Fc domain sufficient to bind CD16 is an Fc domain of a human IgG1 comprising S354C, Q347R, D399V, and F405T substitutions, numbered according to the Kabat numbering.
 63. The protein according to claim 47, wherein the protein comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:189; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:187; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:190, wherein the first polypeptide is linked to the second polypeptide via heterodimerization and at least one disulfide bond, wherein the third polypeptide is linked to the first polypeptide via a disulfide bond.
 64. The protein according to claim 47, wherein the first antigen-binding site comprising a Fab fragment that binds NKG2D, the second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33 and the antibody Fc domain or a portion thereof sufficient to bind CD16 are linked as represented in FIG. 19 .
 65. The protein according to claim 47, wherein the first antigen-binding site comprising a Fab fragment that binds NKG2D, the second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33 and the antibody Fc domain or a portion thereof sufficient to bind CD16 are linked as represented in FIG. 44 .
 66. A method of producing the protein according to claim 47, wherein the method comprises: (a) culturing a host cell under conditions suitable for expression of the protein, wherein the host cell comprises a first nucleic acid encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO:189, a second nucleic acid encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO:187, and a third nucleic acid encoding a third polypeptide comprising the amino acid sequence of SEQ ID NO:190; and (b) isolating and purifying the protein.
 67. A method of treating CD33-expressing cancer comprising administering a therapeutically effective amount of the protein according to claim 47 to a patient in need thereof.
 68. The method according to claim 67, wherein the CD33-expressing cancer is selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), myeloid blast crisis of CIVIL, acute lymphoblastic leukemia (ALL), acute lymphoblastic lymphoma, myeloproliferative neoplasms (MPNs), lymphoma, non-Hodgkin lymphomas, and classical Hodgkin lymphoma.
 69. A protein comprising: (a) a first antigen-binding site comprising a Fab fragment that binds NKG2D, wherein the Fab fragment comprises the amino acid sequence of SEQ ID NO:114, the amino acid sequence of SEQ ID NO:115, the amino acid sequence of SEQ ID NO:116, the amino acid sequence of SEQ ID NO:192, the amino acid sequence of SEQ ID NO:112, and the amino acid sequences of SEQ ID NO:193; (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33, wherein the scFv comprises the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:49, the amino acid sequence of SEQ ID NO:50, the amino acid sequence of SEQ ID NO:181, the amino acid sequence of SEQ ID NO:46, and the amino acid sequences of SEQ ID NO:182; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16.
 70. A pharmaceutical composition comprising a protein and a pharmaceutically acceptable carrier, wherein the protein comprises: (a) a first antigen-binding site comprising a Fab fragment that binds NKG2D, wherein the Fab fragment comprises the amino acid sequence of SEQ ID NO:114, the amino acid sequence of SEQ ID NO:115, the amino acid sequence of SEQ ID NO:116, the amino acid sequence of SEQ ID NO:111, the amino acid sequence of SEQ ID NO:112, and the amino acid sequences of SEQ ID NO:113; (b) a second antigen-binding site comprising a single-chain variable fragment (scFv) that binds CD33, wherein the scFv comprises the amino acid sequence of SEQ ID NO:48, the amino acid sequence of SEQ ID NO:49, the amino acid sequence of SEQ ID NO:50, the amino acid sequence of SEQ ID NO:45, the amino acid sequence of SEQ ID NO:46, and the amino acid sequences of SEQ ID NO:47; and (c) an antibody Fc domain or a portion thereof sufficient to bind CD16.
 71. A nucleic acid encoding: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO:189; (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:187; or (c) a polypeptide comprising the amino acid sequence of SEQ ID NO:190.
 72. The nucleic acid of claim 71, wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:189.
 73. The nucleic acid of claim 71, wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:187.
 74. The nucleic acid of claim 71, wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:190. 